Method and apparatus for continuous analyte monitoring

ABSTRACT

Embodiments of the invention provide analyte sensors and sensor systems such as amperometric glucose sensors used in the management of diabetes as well as optimized methods for monitoring analytes using such sensors and sensor systems.

BACKGROUND OF THE INVENTION

1. Field of the Invention

Analyte sensors (e.g. glucose sensors used in the management ofdiabetes) and methods and materials for making and using such sensors.

2. Description of Related Art

Analyte sensors such as biosensors include devices that use biologicalelements to convert a chemical analyte in a matrix into a detectablesignal. There are many types of biosensors used for a wide variety ofanalytes. The most studied type of biosensor is the amperometric glucosesensor, which is crucial to the successful glucose level control fordiabetes.

A typical glucose sensor works according to the following chemicalreactions:

The glucose oxidase is used to catalyze the reaction between glucose andoxygen to yield gluconic acid and hydrogen peroxide (equation 1). TheH₂O₂ reacts electrochemically as shown in equation 2, and the currentcan be measured by a potentiostat. These reactions, which occur in avariety of oxidoreductases known in the art, are used in a number ofsensor designs.

As analyte sensor technology matures and new applications for sensortechnology are developed, there is a need for methods and materials thatfacilitate the use of sensors in the wide variety of situations in whichthe measurement of an analyte is desirable.

SUMMARY OF THE INVENTION

Embodiments of the invention disclosed herein include analyte sensorsand sensor systems such as amperometric glucose sensors used in themanagement of diabetes as well as optimized methods for monitoringanalytes using such sensors and sensor systems. One embodiment of theinvention is an analyte sensor apparatus comprising: an elongated baselayer; a conductive layer disposed on the base layer and comprising areference electrode, a working electrode and a counter electrode; ananalyte sensing layer disposed on the conductive layer; an analytemodulating layer disposed on the analyte sensing layer, wherein theanalyte modulating layer comprises a composition that modulates thediffusion of an analyte diffusing through the analyte modulating layer;and a cover layer disposed on the analyte sensor apparatus, wherein thecover layer comprises an aperture positioned on the cover layer so as tofacilitate an analyte contacting and diffusing through the analytemodulating layer and contacting the analyte sensing layer. Typicalembodiments of the invention are comprised of biocompatible materialsand/or have structural elements and organizations of elements designedfor implantation within a mammal. Methodological embodiments of theinvention include methods for making and using the sensor embodimentsdisclosed herein. Certain embodiments of the invention include methodsof using a specific sensor element and/or a specific constellation ofsensor elements to produce and/or facilitate one or more properties ofthe sensor embodiments disclosed herein (e.g. sensor initialization andstart-up).

In some embodiments of the invention, an element of the sensor apparatussuch as an electrode or an aperture is designed to have a specificconfiguration and/or is made from a specific material and/or ispositioned relative to the other elements so as to facilitate a functionof the sensor. In one such embodiment of the invention, a workingelectrode, a counter electrode and a reference electrode arepositionally distributed on the base and/or the conductive layer in aconfiguration that facilitates sensor function.

Optionally embodiments of the apparatus comprise a plurality of workingelectrodes and/or counter electrodes and/or reference electrodes (e.g. 3working electrodes, a reference electrode and a counter electrode), inorder to, for example, provide redundant sensing capabilities. Certainembodiments of the invention comprising a single sensor. Otherembodiments of the invention comprise multiple sensors. In someembodiments of the invention, a pulsed voltage is used to obtain asignal from one or more electrodes of a sensor. Optionally, theplurality of working, counter and reference electrodes are configuredtogether as a unit and positionally distributed on the conductive layerin a repeating pattern of units. In certain embodiments of theinvention, the elongated base layer is made from a flexible materialthat allows the sensor to twist and bend when implanted in vivo; and theelectrodes are grouped in a configuration that facilitates an in vivofluid contacting at least one of working electrode as the sensorapparatus twists and bends when implanted in vivo. In some embodiments,the electrodes are grouped in a configuration that allows the sensor tocontinue to function if a portion of the sensor having one or moreelectrodes is dislodged from an in vivo environment and exposed to an exvivo environment.

In embodiments of the invention, a method of sensing an analyte isprovided, comprising applying a first electrode potential to an analytesensor for a first predetermined period of time, applying a secondelectrode potential to the analyte sensor for a second predeterminedperiod of time, repeating the application of the first electrodepotential and the second electrode potential continuously over a sensorduration time period, and receiving and monitoring signals from theanalyte sensor during the sensor duration time period. In furtherembodiments, the sensor in initialized, which can include hydration andrun-in, prior to application of the first electrode potential. Thesensor duration time period preferably is the entire time that thesensor is implanted in the body and being used for analyte sensing. If ahydration and/or other run-in period such as for initialization is used,the sensor duration time period preferably starts after that run-inperiod unless the same first and second electrode potentials are usedduring that period as well. In further embodiments, the voltageswitching of the invention could last for a different period of time,for example, for the time it takes to get one or a predetermined numberof analyte readings. In certain embodiments, the sensor time periodlasts throughout the entire sensing period of a sensor, until the sensoris disconnected from its electronics, physically or by the electronicsbeing otherwise turned off such that no current is being applied to thesensor. The sensor duration time period may be greater than 30 minutes,greater than an hour, greater than 3 hours, or even more time, such as aday.

In further embodiments, a third electrode potential is applied to theanalyte sensor for a third predetermined period of time and theapplication of the first electrode potential, second electrode potentialand third electrode potential are repeated over the sensor duration timeperiod. In further embodiments, additional electrode potentials may beapplied for additional periods of time. In embodiments of the invention,the different electrode potentials are stepped potentials. In preferredembodiments, the electrode potentials are varied continuously over thesensor duration time, whether there be two, three or more differentpotentials used.

In embodiments of the present invention, the concentration of ananalyte, such as glucose, may be calculated from the signals receivedand monitored from the analyte sensor. Calculating the concentration ofthe analyte may include evaluating the overall change in the signalsfrom the analyte sensor during the first predetermined period of timeand/or during the second predetermined period of time. In furtherembodiments, the calculations may include analyzing the relaxationkinetics of the signals from the analyte sensor during the firstpredetermined period of time and/or the second predetermined period oftime. In further embodiments, the calculations may include calculatingthe total charge transfer from signals received from the analyte sensorduring the first and/or second predetermined period of times. If thereare more than two voltages used per cycles, the calculations can be madeduring those periods of times as well.

In embodiments of the present invention, calculating the concentrationof the analyte, or assessing other characteristics of the sensor, anumber of equations may be used. They may also be used to determinewhether the sensor is functioning properly. For example, two possibleequations that may be used are:

${i(t)} = {\frac{{nFADC}_{R}}{d}\left\lbrack {1 + {2{\sum\limits_{n = 1}^{\infty}\;{\mathbb{e}}^{{- \frac{n^{2}\pi^{2}D}{d^{2}}}t}}}} \right\rbrack}$or${i(t)} = {{\frac{{nFADC}_{R}}{d}\left\lbrack {1 + {2{\sum\limits_{n = 1}^{\infty}\;{\mathbb{e}}^{{- \frac{n^{2}\pi^{2}D}{d^{2}}}t}}}} \right\rbrack} + \frac{{nFAD}^{1\text{/}2}C_{0}}{\pi^{1\text{/}2}t^{1\text{/}2}}}$

In addition or as an alternative, analyzing signals received from thesensor can be accomplished using one or more components of the followingequation:

${i(t)} = {{a\frac{{nFADC}_{R}}{d}{\mathbb{e}}^{{- \frac{n^{2}\pi^{2}D}{d^{2}}}t}} + \frac{{nFAD}^{1\text{/}2}C_{0}}{\pi^{1\text{/}2}t^{1\text{/}2}} + {\beta\frac{{nFADC}_{R}}{d}}}$

In embodiments of the present invention, the first predetermined periodof time is 10 seconds. In further embodiments, the second predeterminedperiod of time is about 10 seconds. In other embodiments, the first andsecond predetermined periods of time, as well as any other predeterminedperiods of time in a cycle, may be independently selected from the groupconsisting of 1, 3, 5, 7, 10, 15, 30, 45, 60, 90 and 120 seconds.

In further embodiments, at least one of the first electrode potentialand the second electrode potential is about 535 millivolts. Otherpossible electrode potentials may be, for example, −535, 0, 177, 280,535, 635 or 1.070 millivolts.

Embodiments of the present invention are disclosed that include ananalyte sensing system, the analyte sensing system comprising an analytesensor for implantation in a mammal, and a sensor electronics device incommunication with the analyte sensor, the sensor electronics deviceincluding circuitry to apply a first electrode potential to an analytesensor for a first predetermined period of time, apply a secondelectrode potential to the analyte sensor for a second predeterminedperiod of time, repeat the application of the first electrode potentialand the second electrode potential continuously over a sensor durationtime period; and receive signals from the analyte sensor during thesensor duration time period. In further embodiments, the sensorelectronics device further includes circuitry to initiate the analytesensor prior to the application of the first electrode potential for thefirst predetermined period of time. In further embodiments the sensorelectronics device further includes circuitry to apply a third electrodepotential to the analyte sensor for a third predetermined period of timeand repeat the applicant of the first electrode potential, secondelectrode potential and third electrode potential over the sensorduration time period. In still further embodiments, the first, secondand third electrode potential are stepped electrode potentials.

In further embodiments, the analyte sensing system further comprises amonitoring device in communication with the electronics device, whereinthe monitoring device includes circuitry to monitor the signals receivedfrom the analyte sensor and to calculate the concentration of theanalyte from the signals. The monitoring device may be connecteddirectly to the sensor and/or sensor electronics or may receive datawirelessly. The sensor electronics may be part of the monitor orseparate from the monitor.

In typical embodiments of the invention, the sensor is operativelycoupled to further elements (e.g. electronic components) such aselements designed to transmit and/or receive a signal, monitors,processors and the like as well as devices that can use sensor data tomodulate a patient's physiology such as medication infusion pumps. Forexample, in some embodiments of the invention, the sensor is operativelycoupled to a sensor input capable of receiving a signal from the sensorthat is based on a sensed physiological characteristic value in themammal; and a processor coupled to the sensor input, wherein theprocessor is capable of characterizing one or more signals received fromthe sensor. A wide variety of sensor configurations as disclosed hereincan be used in such systems. Optionally, for example, the sensorcomprises three working electrodes, one counter electrode and onereference electrode. In certain embodiments, at least one workingelectrode is coated with an analyte sensing layer comprising glucoseoxidase and at least one working electrode is not coated with an analytesensing layer comprising glucose oxidase.

Other objects, features and advantages of the present invention willbecome apparent to those skilled in the art from the following detaileddescription. It is to be understood, however, that the detaileddescription and specific examples, while indicating some embodiments ofthe present invention are given by way of illustration and notlimitation. Many changes and modifications within the scope of thepresent invention may be made without departing from the spirit thereof,and the invention includes all such modifications.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides a schematic of the well known reaction between glucoseand glucose oxidase. As shown in a stepwise manner, this reactioninvolves glucose oxidase (GOx), glucose and oxygen in water. In thereductive half of the reaction, two protons and electrons aretransferred from 1-D-glucose to the enzyme yielding d-gluconolactone. Inthe oxidative half of the reaction, the enzyme is oxidized by molecularoxygen yielding hydrogen peroxide. The d-gluconolactone then reacts withwater to hydrolyze the lactone ring and produce gluconic acid. Incertain electrochemical sensors of the invention, the hydrogen peroxideproduced by this reaction is oxidized at the working electrode(H₂O₂→2H++O₂+2e⁻).

FIG. 2 provides a diagrammatic view of a typical layered analyte sensorconfiguration of the current invention.

FIG. 3 provides a perspective view illustrating a subcutaneous sensorinsertion set, a telemetered characteristic monitor transmitter device,and a data receiving device embodying features of the invention.

FIGS. 4A and 4B provide graphs of the current readings (Isigs) via timeas taken during experiments using certain embodiments of the invention.

FIGS. 5A and 5B provide graphs illustrating properties for lineardiffusion in a simple electrode system according to embodiments of theinvention.

FIGS. 6A-6C provide graphs showing waveforms fitted using an equationaccording to embodiments of the invention. FIG. 6A shows measuredcurrent from a first and second voltage. FIG. 6B shows the measured andcalculated currents for the first voltage and FIG. 6C shows the measuredand calculated currents for the second voltage.

DETAILED DESCRIPTION OF THE EMBODIMENTS

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted. A number of termsare defined below.

All publications mentioned herein are incorporated herein by referenceto disclose and describe the methods and/or materials in connection withwhich the publications are cited. Publications cited herein are citedfor their disclosure prior to the filing date of the presentapplication. Nothing here is to be construed as an admission that theinventors are not entitled to antedate the publications by virtue of anearlier priority date or prior date of invention. Further the actualpublication dates may be different from those shown and requireindependent verification.

Before the present compositions and methods etc. are described, it is tobe understood that this invention is not limited to the particularmethodology, protocol and reagent described as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to limit the scope of the present invention which will belimited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a”, “and”, and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “anoxidoreductase” includes a plurality of such oxidoreductases andequivalents thereof known to those skilled in the art, and so forth. Allnumbers recited in the specification and associated claims that refer tovalues that can be numerically characterized with a value other than awhole number (e.g. the concentration of a compound in a solution) areunderstood to be modified by the term “about”.

The term “analyte” as used herein is a broad term and is used in itsordinary sense, including, without limitation, to refer to a substanceor chemical constituent in a fluid such as a biological fluid (forexample, blood, interstitial fluid, cerebral spinal fluid, lymph fluidor urine) that can be analyzed. Analytes can include naturally occurringsubstances, artificial substances, metabolites, and/or reactionproducts. In some embodiments, the analyte for measurement by thesensing regions, devices, and methods is glucose. However, otheranalytes are contemplated as well, including but not limited to,lactate. Salts, sugars, proteins fats, vitamins and hormones naturallyoccurring in blood or interstitial fluids can constitute analytes incertain embodiments. The analyte can be naturally present in thebiological fluid or endogenous; for example, a metabolic product, ahormone, an antigen, an antibody, and the like. Alternatively, theanalyte can be introduced into the body or exogenous, for example, acontrast agent for imaging, a radioisotope, a chemical agent, afluorocarbon-based synthetic blood, or a drug or pharmaceuticalcomposition, including but not limited to insulin. The metabolicproducts of drugs and pharmaceutical compositions are also contemplatedanalytes.

The term “oxidoreductase” is used according to its art accepted meaning,i.e. an enzyme that catalyzes the transfer of electrons from onemolecule (the reductant, also called the hydrogen or electron donor) toanother (the oxidant, also called the hydrogen or electron acceptor).Typical oxidoreductases include glucose oxidase and lactate oxidase. Theterm “carrier polypeptide” or “carrier protein” is used according to itsart accepted meaning of an additive included to maintain the stabilityof a polypeptide, for example the ability of an oxidoreductasepolypeptide to maintain certain qualitative features such as physicaland chemical properties (e.g. an ability to oxidize glucose) of acomposition comprising a polypeptide for a period of time. A typicalcarrier protein commonly used in the art is albumin.

The term “sensor,” as used herein, is a broad term and is used in itsordinary sense, including, without limitation, the portion or portionsof an analyte-monitoring device that detects an analyte. In oneembodiment, the sensor includes an electrochemical cell that has aworking electrode, a reference electrode, and optionally a counterelectrode passing through and secured within the sensor body forming anelectrochemically reactive surface at one location on the body, anelectronic connection at another location on the body, and a membranesystem affixed to the body and covering the electrochemically reactivesurface. During general operation of the sensor, a biological sample(for example, blood or interstitial fluid), or a portion thereof,contacts (directly or after passage through one or more membranes ordomains) an enzyme (for example, glucose oxidase); the reaction of thebiological sample (or portion thereof results in the formation ofreaction products that allow a determination of the analyte level in thebiological sample.

The terms “electrochemically reactive surface” and “electroactivesurface” as used herein are broad terms and are used in their ordinarysense, including, without limitation, the surface of an electrode wherean electrochemical reaction takes place. In one example, a workingelectrode measures hydrogen peroxide produced by the enzyme catalyzedreaction of the analyte being detected reacts creating an electriccurrent (for example, detection of glucose analyte utilizing glucoseoxidase produces H₂O₂ as a by product, H₂O₂ reacts with the surface ofthe working electrode producing two protons (2H⁺), two electrons (2e⁻)and one molecule of oxygen (O₂) which produces the electronic currentbeing detected). In the case of the counter electrode, a reduciblespecies, for example, O₂ is reduced at the electrode surface in order tobalance the current being generated by the working electrode.

The term “sensing region” as used herein is a broad term and is used inits ordinary sense, including, without limitation, the region of amonitoring device responsible for the detection of a particular analyte.In an illustrative embodiment, the sensing region can comprise anon-conductive body, a working electrode, a reference electrode, and acounter electrode passing through and secured within the body formingelectrochemically reactive surfaces on the body and an electronicconnective means at another location on the body, and a one or morelayers covering the electrochemically reactive surface.

The terms “electrical potential” and “potential” as used herein, arebroad terms and are used in their ordinary sense, including, withoutlimitation, the electrical potential difference between two points in acircuit which is the cause of the flow of a current. The term “systemnoise,” as used herein, is a broad term and is used in its ordinarysense, including, without limitation, unwanted electronic ordiffusion-related noise which can include Gaussian, motion-related,flicker, kinetic, or other white noise, for example.

The terms “interferents” and “interfering species,” as used herein, arebroad terms and are used in their ordinary sense, including, but notlimited to, effects and/or chemical species/compounds that interferewith the measurement of an analyte of interest in a sensor to produce asignal that does not accurately represent the analyte measurement. Inone example of an electrochemical sensor, interfering species arecompounds with an oxidation potential that overlaps with the analyte tobe measured.

As discussed in detail below, embodiments of the invention relate to theuse of an electrochemical sensor that measures a concentration of ananalyte of interest or a substance indicative of the concentration orpresence of the analyte in fluid. In some embodiments, the sensor is acontinuous device, for example a subcutaneous, transdermal, orintravascular device. In some embodiments, the device can analyze aplurality of intermittent blood samples. The sensor embodimentsdisclosed herein can use any known method, including invasive, minimallyinvasive, and non-invasive sensing techniques, to provide an outputsignal indicative of the concentration of the analyte of interest.Typically, the sensor is of the type that senses a product or reactantof an enzymatic reaction between an analyte and an enzyme in thepresence of oxygen as a measure of the analyte in vivo or in vitro. Suchsensors typically comprise a membrane surrounding the enzyme throughwhich an analyte migrates. The product is then measured usingelectrochemical methods and thus the output of an electrode systemfunctions as a measure of the analyte. In some embodiments, the sensorcan use an amperometric, coulometric, conductimetric, and/orpotentiometric technique for measuring the analyte.

Embodiments of the invention disclosed herein provide sensors of thetype used, for example, in subcutaneous or transcutaneous monitoring ofblood glucose levels in a diabetic patient. A variety of implantable,electrochemical biosensors have been developed for the treatment ofdiabetes and other life-threatening diseases. Many existing sensordesigns use some form of immobilized enzyme to achieve theirbio-specificity. Embodiments of the invention described herein can beadapted and implemented with a wide variety of known electrochemicalsensors, including for example, U.S. Patent Application No. 20050115832,U.S. Pat. Nos. 6,001,067, 6,702,857, 6,212,416, 6,119,028, 6,400,974,6,595,919, 6,141,573, 6,122,536, 6,512,939 5,605,152, 4,431,004,4,703,756, 6,514,718, 5,985,129, 5,390,691, 5,391,250, 5,482,473,5,299,571, 5,568,806, 5,494,562, 6,120,676, 6,542,765 as well as PCTInternational Publication Numbers WO 01/58348, WO 04/021877, WO03/034902, WO 03/035117, WO 03/035891, WO 03/023388, WO 03/022128, WO03/022352, WO 03/023708, WO 03/036255, WO03/036310 WO 08/042,625, and WO03/074107, and European Patent Application EP 1153571, the contents ofeach of which are incorporated herein by reference.

As discussed in detail below, embodiments of the invention disclosedherein provide sensor elements having enhanced material propertiesand/or architectural configurations and sensor systems (e.g. thosecomprising a sensor and associated electronic components such as amonitor, a processor and the like) constructed to include such elements.The disclosure further provides methods for making and using suchsensors and/or architectural configurations. While some embodiments ofthe invention pertain to glucose and/or lactate sensors, a variety ofthe elements disclosed herein (e.g. electrodes and electrode designs)can be adapted for use with any one of the wide variety of sensors knownin the art. The analyte sensor elements, architectures and methods formaking and using these elements that are disclosed herein can be used toestablish a variety of layered sensor structures. Such sensors of theinvention exhibit a surprising degree of flexibility and versatility,characteristics which allow a wide variety of sensor configurations tobe designed to examine a wide variety of analyte species.

In typical embodiments of the present invention, the transduction of theanalyte concentration into a processable signal is by electrochemicalmeans. These transducers may include any of a wide variety ofamperometric, potentiometric, or conductimetric base sensors known inthe art. Moreover, the microfabrication sensor techniques and materialsof the instant invention may be applied to other types of transducers(e.g., acoustic wave sensing devices, thermistors, gas-sensingelectrodes, field-effect transistors, optical and evanescent field waveguides, and the like) fabricated in a substantially nonplanar, oralternatively, a substantially planar manner. A useful discussion andtabulation of transducers which may be exploited in a biosensor as wellas the kinds of analytical applications in which each type of transduceror biosensor, in general, may be utilized, is found in an article byChristopher R. Lowe in Trends in Biotech. 1984, 2(3), 59-65.

Specific aspects of embodiments of the invention are discussed in detailin the following sections.

I. Typical Elements, Configurations and Analyte Sensor Embodiments ofthe Invention

A. Typical Architectures Found in of Embodiments of the Invention

FIG. 2 illustrates a cross-section of a typical sensor embodiment 100 ofthe present invention. This sensor embodiment is formed from a pluralityof components that are typically in the form of layers of variousconductive and non-conductive constituents disposed on each otheraccording to art accepted methods and/or the specific methods of theinvention disclosed herein. The components of the sensor are typicallycharacterized herein as layers because, for example, it allows for afacile characterization of the sensor structure shown in FIG. 2.Artisans will understand however, that in certain embodiments of theinvention, the sensor constituents are combined such that multipleconstituents form one or more heterogeneous layers. In this context,those of skill in the art understand that the ordering of the layeredconstituents can be altered in various embodiments of the invention.

The embodiment shown in FIG. 2 includes a base layer 102 to support thesensor 100. The base layer 102 can be made of a material such as a metaland/or a ceramic and/or a polymeric substrate, which may beself-supporting or further supported by another material as is known inthe art. Embodiments of the invention include a conductive layer 104which is disposed on and/or combined with the base layer 102. Typicallythe conductive layer 104 comprises one or more electrodes. An operatingsensor 100 typically includes a plurality of electrodes such as aworking electrode, a counter electrode and a reference electrode. Otherembodiments may also include a plurality of working and/or counterand/or reference electrodes and/or one or more electrodes that performsmultiple functions, for example one that functions as both as areference and a counter electrode.

As discussed in detail below, the base layer 102 and/or conductive layer104 can be generated using many known techniques and materials. Incertain embodiments of the invention, the electrical circuit of thesensor is defined by etching the disposed conductive layer 104 into adesired pattern of conductive paths. A typical electrical circuit forthe sensor 100 comprises two or more adjacent conductive paths withregions at a proximal end to form contact pads and regions at a distalend to form sensor electrodes. An electrically insulating cover layer106 such as a polymer coating can be disposed on portions of the sensor100. Acceptable polymer coatings for use as the insulating protectivecover layer 106 can include, but are not limited to, non-toxicbiocompatible polymers such as silicone compounds, polyimides,biocompatible solder masks, epoxy acrylate copolymers, or the like. Inthe sensors of the present invention, one or more exposed regions orapertures 108 can be made through the cover layer 106 to open theconductive layer 104 to the external environment and to, for example,allow an analyte such as glucose to permeate the layers of the sensorand be sensed by the sensing elements. Apertures 108 can be formed by anumber of techniques, including laser ablation, tape masking, chemicalmilling or etching or photolithographic development or the like. Incertain embodiments of the invention, during manufacture, a secondaryphotoresist can also be applied to the protective layer 106 to definethe regions of the protective layer to be removed to form theaperture(s) 108. The exposed electrodes and/or contact pads can alsoundergo secondary processing (e.g. through the apertures 108), such asadditional plating processing, to prepare the surfaces and/or strengthenthe conductive regions.

In the sensor configuration shown in FIG. 2, an analyte sensing layer110 (which is typically a sensor chemistry layer, meaning that materialsin this layer undergo a chemical reaction to produce a signal that canbe sensed by the conductive layer) is disposed on one or more of theexposed electrodes of the conductive layer 104. Typically, the analytesensing layer 110 is an enzyme layer. Most typically, the analytesensing layer 110 comprises an enzyme capable of producing and/orutilizing oxygen and/or hydrogen peroxide, for example the enzymeglucose oxidase. Optionally the enzyme in the analyte sensing layer iscombined with a second carrier protein such as human serum albumin,bovine serum albumin or the like. In an illustrative embodiment, anoxidoreductase enzyme such as glucose oxidase in the analyte sensinglayer 110 reacts with glucose to produce hydrogen peroxide, a compoundwhich then modulates a current at an electrode. As this modulation ofcurrent depends on the concentration of hydrogen peroxide, and theconcentration of hydrogen peroxide correlates to the concentration ofglucose, the concentration of glucose can be determined by monitoringthis modulation in the current. In a specific embodiment of theinvention, the hydrogen peroxide is oxidized at a working electrodewhich is an anode (also termed herein the anodic working electrode),with the resulting current being proportional to the hydrogen peroxideconcentration. Such modulations in the current caused by changinghydrogen peroxide concentrations can by monitored by any one of avariety of sensor detector apparatuses such as a universal sensoramperometric biosensor detector or one of the other variety of similardevices known in the art such as glucose monitoring devices produced byMedtronic MiniMed.

In embodiments of the invention, the analyte sensing layer 110 can beapplied over portions of the conductive layer or over the entire regionof the conductive layer. Typically the analyte sensing layer 110 isdisposed on the working electrode which can be the anode or the cathode.Optionally, the analyte sensing layer 110 is also disposed on a counterand/or reference electrode. While the analyte sensing layer 110 can beup to about 1000 microns (μm) in thickness, typically the analytesensing layer is relatively thin as compared to those found in sensorspreviously described in the art, and is for example, typically less than1, 0.5, 0.25 or 0.1 microns in thickness. As discussed in detail below,some methods for generating a thin analyte sensing layer 110 includebrushing the layer onto a substrate (e.g. the reactive surface of aplatinum black electrode), as well as spin coating processes, dip anddry processes, low shear spraying processes, ink jet printing processes,silk screen processes and the like. In certain embodiments of theinvention, brushing is used to: (1) allow for a precise localization ofthe layer; and (2) push the layer deep into the architecture of thereactive surface of an electrode (e.g. platinum black produced by anelectrodeposition process).

Typically, the analyte sensing layer 110 is coated and or disposed nextto one or more additional layers. Optionally, the one or more additionallayers includes a protein layer 116 disposed upon the analyte sensinglayer 110. Typically, the protein layer 116 comprises a protein such ashuman serum albumin, bovine serum albumin or the like. Typically, theprotein layer 116 comprises human serum albumin. In some embodiments ofthe invention, an additional layer includes an analyte modulating layer112 that is disposed above the analyte sensing layer 110 to regulateanalyte contact with the analyte sensing layer 110. For example, theanalyte modulating membrane layer 112 can comprise a glucose limitingmembrane, which regulates the amount of glucose that contacts an enzymesuch as glucose oxidase that is present in the analyte sensing layer.Such glucose limiting membranes can be made from a wide variety ofmaterials known to be suitable for such purposes, e.g., siliconecompounds such as polydimethyl siloxanes, polyurethanes, polyureacellulose acetates, Nafion, polyester sulfonic acids (e.g. Kodak AQ),hydrogels or any other suitable hydrophilic membranes known to thoseskilled in the art.

In typical embodiments of the invention, an adhesion promoter layer 114is disposed between the analyte modulating layer 112 and the analytesensing layer 110 as shown in FIG. 2 in order to facilitate theircontact and/or adhesion. In a specific embodiment of the invention, anadhesion promoter layer 114 is disposed between the analyte modulatinglayer 112 and the protein layer 116 as shown in FIG. 2 in order tofacilitate their contact and/or adhesion. The adhesion promoter layer114 can be made from any one of a wide variety of materials known in theart to facilitate the bonding between such layers. Typically, theadhesion promoter layer 114 comprises a silane compound. In alternativeembodiments, protein or like molecules in the analyte sensing layer 110can be sufficiently crosslinked or otherwise prepared to allow theanalyte modulating membrane layer 112 to be disposed in direct contactwith the analyte sensing layer 110 in the absence of an adhesionpromoter layer 114.

In certain embodiments of the invention, a sensor is designed to includeadditional layers such as an interference rejection layer discussedbelow.

B. Typical Analyte Sensor Constituents Used in Embodiments of theInvention

The following disclosure provides examples of typicalelements/constituents used in sensor embodiments of the invention. Whilethese elements can be described as discreet units (e.g. layers), thoseof skill in the art understand that sensors can be designed to containelements having a combination of some or all of the material propertiesand/or functions of the elements/constituents discussed below (e.g. anelement that serves both as a supporting base constituent and/or aconductive constituent and/or a matrix for the analyte sensingconstituent and which further functions as an electrode in the sensor).Those in the art understand that these thin film analyte sensors can beadapted for use in a number of sensor systems such as those describedbelow.

Base Constituent

Sensors of the invention typically include a base constituent (see, e.g.element 102 in FIG. 2). The term “base constituent” is used hereinaccording to art accepted terminology and refers to the constituent inthe apparatus that typically provides a supporting matrix for theplurality of constituents that are stacked on top of one another andcomprise the functioning sensor. In one form, the base constituentcomprises a thin film sheet of insulative (e.g. electrically insulativeand/or water impermeable) material. This base constituent can be made ofa wide variety of materials having desirable qualities such asdielectric properties, water impermeability and hermeticity. Somematerials include metallic, and/or ceramic and/or polymeric substratesor the like.

The base constituent may be self-supporting or further supported byanother material as is known in the art. In one embodiment of the sensorconfiguration shown in FIG. 2, the base constituent 102 comprises aceramic. Alternatively, the base constituent comprises a polymericmaterial such as a polyimmide. In an illustrative embodiment, theceramic base comprises a composition that is predominantly Al₂O₃ (e.g.96%). The use of alumina as an insulating base constituent for use withimplantable devices is disclosed in U.S. Pat. Nos. 4,940,858, 4,678,868and 6,472,122 which are incorporated herein by reference. The baseconstituents of the invention can further include other elements knownin the art, for example hermetical vias (see, e.g. WO 03/023388).Depending upon the specific sensor design, the base constituent can berelatively thick constituent (e.g. thicker than 50, 100, 200, 300, 400,500 or 1000 microns). Alternatively, one can utilize a nonconductiveceramic, such as alumina, in thin constituents, e.g., less than about 30microns.

Conductive Constituent

The electrochemical sensors of the invention typically include aconductive constituent disposed upon the base constituent that includesat least one electrode for contacting an analyte or its byproduct (e.g.oxygen and/or hydrogen peroxide) to be assayed (see, e.g. element 104 inFIG. 2). The term “conductive constituent” is used herein according toart accepted terminology and refers to electrically conductive sensorelements such as electrodes which are capable of measuring and adetectable signal and conducting this to a detection apparatus. Anillustrative example of this is a conductive constituent that canmeasure an increase or decrease in current in response to exposure to astimuli such as the change in the concentration of an analyte or itsbyproduct as compared to a reference electrode that does not experiencethe change in the concentration of the analyte, a coreactant (e.g.oxygen) used when the analyte interacts with a composition (e.g. theenzyme glucose oxidase) present in analyte sensing constituent 110 or areaction product of this interaction (e.g. hydrogen peroxide).Illustrative examples of such elements include electrodes which arecapable of producing variable detectable signals in the presence ofvariable concentrations of molecules such as hydrogen peroxide oroxygen. Typically one of these electrodes in the conductive constituentis a working electrode, which can be made from non-corroding metal orcarbon. A carbon working electrode may be vitreous or graphitic and canbe made from a solid or a paste. A metallic working electrode may bemade from platinum group metals, including palladium or gold, or anon-corroding metallically conducting oxide, such as ruthenium dioxide.Alternatively the electrode may comprise a silver/silver chlorideelectrode composition. The working electrode may be a wire or a thinconducting film applied to a substrate, for example, by coating orprinting. Typically, only a portion of the surface of the metallic orcarbon conductor is in electrolytic contact with the analyte-containingsolution. This portion is called the working surface of the electrode.The remaining surface of the electrode is typically isolated from thesolution by an electrically insulating cover constituent 106. Examplesof useful materials for generating this protective cover constituent 106include polymers such as polyimides, polytetrafluoroethylene,polyhexafluoropropylene and silicones such as polysiloxanes.

In addition to the working electrode, the analyte sensors of theinvention typically include a reference electrode or a combinedreference and counter electrode (also termed a quasi-reference electrodeor a counter/reference electrode). If the sensor does not have acounter/reference electrode then it may include a separate counterelectrode, which may be made from the same or different materials as theworking electrode. Typical sensors of the present invention have one ormore working electrodes and one or more counter, reference, and/orcounter/reference electrodes. One embodiment of the sensor of thepresent invention has two, three or four or more working electrodes.These working electrodes in the sensor may be integrally connected orthey may be kept separate.

Typically for in vivo use, embodiments of the present invention areimplanted subcutaneously in the skin of a mammal for direct contact withthe body fluids of the mammal, such as blood. Alternatively the sensorscan be implanted into other regions within the body of a mammal such asin the intraperotineal space. When multiple working electrodes are used,they may be implanted together or at different positions in the body.The counter, reference, and/or counter/reference electrodes may also beimplanted either proximate to the working electrode(s) or at otherpositions within the body of the mammal. Embodiments of the inventioninclude sensors comprising electrodes constructed from nanostructuredmaterials. As used herein, a “nanostructured material” is an objectmanufactured to have at least one dimension smaller than 100 nm.Examples include, but are not limited to, single-walled nanotubes,double-walled nanotubes, multi-walled nanotubes, bundles of nanotubes,fullerenes, cocoons, nanowires, nanofibres, onions and the like.

Interference Rejection Constituent

The electrochemical sensors of the invention optionally include aninterference rejection constituent disposed between the surface of theelectrode and the environment to be assayed. In particular, certainsensor embodiments rely on the oxidation and/or reduction of hydrogenperoxide generated by enzymatic reactions on the surface of a workingelectrode at a constant potential applied. Because amperometricdetection based on direct oxidation of hydrogen peroxide requires arelatively high oxidation potential, sensors employing this detectionscheme may suffer interference from oxidizable species that are presentin biological fluids such as ascorbic acid, uric acid and acetaminophen.In this context, the term “interference rejection constituent” is usedherein according to art accepted terminology and refers to a coating ormembrane in the sensor that functions to inhibit spurious signalsgenerated by such oxidizable species which interfere with the detectionof the signal generated by the analyte to be sensed. Certaininterference rejection constituents function via size exclusion (e.g. byexcluding interfering species of a specific size). Examples ofinterference rejection constituents include one or more layers orcoatings of compounds such as hydrophilic polyurethanes, celluloseacetate (including cellulose acetate incorporating agents such aspoly(ethylene glycol), polyethersulfones, polytetra-fluoroethylenes, theperfluoronated ionomer Nafion™, polyphenylenediamine, epoxy and thelike. Illustrative discussions of such interference rejectionconstituents are found for example in Ward et al., Biosensors andBioelectronics 17 (2002) 181-189 and Choi et al., Analytical ChimicaActa 461 (2002) 251-260 which are incorporated herein by reference.Other interference rejection constituents include for example thoseobserved to limit the movement of compounds based upon a molecularweight range, for example cellulose acetate as disclosed for example inU.S. Pat. No. 5,755,939, the contents of which are incorporated byreference. Interference rejection membranes useful with embodiments ofthe invention are also described, for example, in U.S. patentapplication Ser. No. 12/572,087, the contents of which are incorporatedby reference. Optionally, the interference rejection membrane comprisescrosslinked methacrylate polymers or crosslinked primary amine polymers.In certain embodiments of the invention, the crosslinked methacrylatepolymers comprise Poly(2-hydroxyethyl methacrylate) (pHEMA) polymershaving an average molecular weight of between 100 and 1000 kilodaltons.Typically the polymers are crosslinked by a hydrophilic crosslinkingagent.

Analyte Sensing Constituent

The electrochemical sensors of the invention include an analyte sensingconstituent disposed on the electrodes of the sensor (see, e.g. element110 in FIG. 2). The term “analyte sensing constituent” is used hereinaccording to art accepted terminology and refers to a constituentcomprising a material that is capable of recognizing or reacting with ananalyte whose presence is to be detected by the analyte sensorapparatus. Typically this material in the analyte sensing constituentproduces a detectable signal after interacting with the analyte to besensed, typically via the electrodes of the conductive constituent. Inthis regard the analyte sensing constituent and the electrodes of theconductive constituent work in combination to produce the electricalsignal that is read by an apparatus associated with the analyte sensor.Typically, the analyte sensing constituent comprises an oxidoreductaseenzyme capable of reacting with and/or producing a molecule whose changein concentration can be measured by measuring the change in the currentat an electrode of the conductive constituent (e.g. oxygen and/orhydrogen peroxide), for example the enzyme glucose oxidase. An enzymecapable of producing a molecule such as hydrogen peroxide can bedisposed on the electrodes according to a number of processes known inthe art. The analyte sensing constituent can coat all or a portion ofthe various electrodes of the sensor. In this context, the analytesensing constituent may coat the electrodes to an equivalent degree.Alternatively the analyte sensing constituent may coat differentelectrodes to different degrees, with for example the coated surface ofthe working electrode being larger than the coated surface of thecounter and/or reference electrode.

Typical sensor embodiments of this element of the invention utilize anenzyme (e.g. glucose oxidase) that has been combined with a secondprotein (e.g. albumin) in a fixed ratio (e.g. one that is typicallyoptimized for glucose oxidase stabilizing properties) and then appliedon the surface of an electrode to form a thin enzyme constituent. In atypical embodiment, the analyte sensing constituent comprises a GOx andHSA mixture. In a typical embodiment of an analyte sensing constituenthaving GOx, the GOx reacts with glucose present in the sensingenvironment (e.g. the body of a mammal) and generates hydrogen peroxideaccording to the reaction shown in FIG. 1, wherein the hydrogen peroxideso generated is anodically detected at the working electrode in theconductive constituent.

As noted above, the enzyme and the second protein (e.g. an albumin) aretypically treated to form a crosslinked matrix (e.g. by adding across-linking agent to the protein mixture). As is known in the art,crosslinking conditions may be manipulated to modulate factors such asthe retained biological activity of the enzyme, its mechanical and/oroperational stability. Illustrative crosslinking procedures aredescribed in U.S. patent application Ser. No. 10/335,506 and PCTpublication WO 03/035891 which are incorporated herein by reference. Forexample, an amine cross-linking reagent, such as, but not limited to,glutaraldehyde, can be added to the protein mixture. The addition of across-linking reagent to the protein mixture creates a protein paste.The concentration of the cross-linking reagent to be added may varyaccording to the concentration of the protein mixture. Whileglutaraldehyde is an illustrative crosslinking reagent, othercross-linking reagents may also be used or may be used in place ofglutaraldehyde. Other suitable cross-linkers also may be used, as willbe evident to those skilled in the art.

The GOx and/or carrier protein concentration may vary for differentembodiments of the invention. For example, the GOx concentration may bewithin the range of approximately 50 mg/ml (approximately 10,000 U/ml)to approximately 700 mg/ml (approximately 150,000 U/ml). Typically theGOx concentration is about 115 mg/ml (approximately 22,000 U/ml). Insuch embodiments, the HSA concentration may vary between about 0.5%-30%(w/v), depending on the GOx concentration. Typically the HSAconcentration is about 1-10% w/v, and most typically is about 5% w/v. Inalternative embodiments of the invention, collagen or BSA or otherstructural proteins used in these contexts can be used instead of or inaddition to HSA. Although GOx is discussed as an illustrative enzyme inthe analyte sensing constituent, other proteins and/or enzymes may alsobe used or may be used in place of GOx, including, but not limited toglucose dehydrogenase or hexokinase, hexose oxidase, lactate oxidase,and the like. Other proteins and/or enzymes may also be used, as will beevident to those skilled in the art. Moreover, although HSA is employedin the example embodiment, other structural proteins, such as BSA,collagens or the like, could be used instead of or in addition to HSA.

As noted above, in some embodiments of the invention, the analytesensing constituent includes a composition (e.g. glucose oxidase)capable of producing a signal (e.g. a change in oxygen and/or hydrogenperoxide concentrations) that can be sensed by the electricallyconductive elements (e.g. electrodes which sense changes in oxygenand/or hydrogen peroxide concentrations). However, other useful analytesensing constituents can be formed from any composition that is capableof producing a detectable signal that can be sensed by the electricallyconductive elements after interacting with a target analyte whosepresence is to be detected. In some embodiments, the compositioncomprises an enzyme that modulates hydrogen peroxide concentrations uponreaction with an analyte to be sensed. Alternatively, the compositioncomprises an enzyme that modulates oxygen concentrations upon reactionwith an analyte to be sensed. In this context, a wide variety of enzymesthat either use or produce hydrogen peroxide and/or oxygen in a reactionwith a physiological analyte are known in the art and these enzymes canbe readily incorporated into the analyte sensing constituentcomposition. A variety of other enzymes known in the art can produceand/or utilize compounds whose modulation can be detected byelectrically conductive elements such as the electrodes that areincorporated into the sensor designs described herein. Such enzymesinclude for example, enzymes specifically described in Table 1, pages15-29 and/or Table 18, pages 111-112 of Protein Immobilization:Fundamentals and Applications (Bioprocess Technology, Vol 14) by RichardF. Taylor (Editor) Publisher: Marcel Dekker; Jan. 7, 1991) the entirecontents of which are incorporated herein by reference.

Other useful analyte sensing constituents can be formed to includeantibodies whose interaction with a target analyte is capable ofproducing a detectable signal that can be sensed by the electricallyconductive elements after interacting with the target analyte whosepresence is to be detected. For example U.S. Pat. No. 5,427,912 (whichis incorporated herein by reference) describes an antibody-basedapparatus for electrochemically determining the concentration of ananalyte in a sample. In this device, a mixture is formed which includesthe sample to be tested, an enzyme-acceptor polypeptide, an enzyme-donorpolypeptide linked to an analyte analog (enzyme-donor polypeptideconjugate), a labeled substrate, and an antibody specific for theanalyte to be measured. The analyte and the enzyme-donor polypeptideconjugate competitively bind to the antibody. When the enzyme-donorpolypeptide conjugate is not bound to antibody, it will spontaneouslycombine with the enzyme acceptor polypeptide to form an active enzymecomplex. The active enzyme then hydrolyzes the labeled substrate,resulting in the generation of an electroactive label, which can then beoxidized at the surface of an electrode. A current resulting from theoxidation of the electroactive compound can be measured and correlatedto the concentration of the analyte in the sample. U.S. Pat. No.5,149,630 (which is incorporated herein by reference) describes anelectrochemical specific binding assay of a ligand (e.g., antigen,hapten or antibody) wherein at least one of the components isenzyme-labelled, and which includes the step of determining the extentto which the transfer of electrons between the enzyme substrate and anelectrode, associated with the substrate reaction, is perturbed bycomplex formation or by displacement of any ligand complex relative tounbound enzyme-labelled component. U.S. Pat. No. 6,410,251 (which isincorporated herein by reference) describes an apparatus and method fordetecting or assaying one constituting member in a specific bindingpair; for example, the antigen in an antigen/antibody pair, by utilizingspecific binding such as binding between an antigen and an antibody,together with redox reaction for detecting a label, wherein an oxygenmicro-electrode with a sensing surface area is used. In addition, U.S.Pat. No. 4,402,819 (which is incorporated herein by reference) describesan antibody-selective potentiometric electrode for the quantitativedetermination of antibodies (as the analyte) in dilute liquid serumsamples employing an insoluble membrane incorporating an antigen havingbonded thereto an ion carrier effecting the permeability of preselectedcations therein, which permeability is a function of specific antibodyconcentrations in analysis, and the corresponding method of analysis.For related disclosures, see also U.S. Pat. Nos. 6,703,210, 5,981,203,5,705,399 and 4,894,253, the contents of which are incorporated hereinby reference.

In addition to enzymes and antibodies, other exemplary materials for usein the analyte sensing constituents of the sensors disclosed hereininclude polymers that bind specific types of cells or cell components(e.g. polypeptides, carbohydrates and the like); single-strand DNA;antigens and the like. The detectable signal can be, for example, anoptically detectable change, such as a color change or a visibleaccumulation of the desired analyte (e.g., cells). Sensing elements canalso be formed from materials that are essentially non-reactive (i.e.,controls). The foregoing alternative sensor elements are beneficiallyincluded, for example, in sensors for use in cell-sorting assays andassays for the presence of pathogenic organisms, such as viruses (HIV,hepatitis-C, etc.), bacteria, protozoa and the like.

Also contemplated are analyte sensors that measure an analyte that ispresent in the external environment and that can in itself produce ameasurable change in current at an electrode. In sensors measuring suchanalytes, the analyte sensing constituent can be optional.

Protein Constituent

The electrochemical sensors of the invention optionally include aprotein constituent disposed between the analyte sensing constituent andthe analyte modulating constituent (see, e.g. element 116 in FIG. 2).The term “protein constituent” is used herein according to art acceptedterminology and refers to constituent containing a carrier protein orthe like that is selected for compatibility with the analyte sensingconstituent and/or the analyte modulating constituent. In typicalembodiments, the protein constituent comprises an albumin such as humanserum albumin. The HSA concentration may vary between about 0.5%-30%(w/v). Typically the HSA concentration is about 1-10% w/v, and mosttypically is about 5% w/v. In alternative embodiments of the invention,collagen or BSA or other structural proteins used in these contexts canbe used instead of or in addition to HSA. This constituent is typicallycrosslinked on the analyte sensing constituent according to art acceptedprotocols.

Adhesion Promoting Constituent

The electrochemical sensors of the invention can include one or moreadhesion promoting (AP) constituents (see, e.g. element 114 in FIG. 2).The term “adhesion promoting constituent” is used herein according toart accepted terminology and refers to a constituent that includesmaterials selected for their ability to promote adhesion betweenadjoining constituents in the sensor. Typically, the adhesion promotingconstituent is disposed between the analyte sensing constituent and theanalyte modulating constituent. Typically, the adhesion promotingconstituent is disposed between the optional protein constituent and theanalyte modulating constituent. The adhesion promoter constituent can bemade from any one of a wide variety of materials known in the art tofacilitate the bonding between such constituents and can be applied byany one of a wide variety of methods known in the art. Typically, theadhesion promoter constituent comprises a silane compound such asγ-aminopropyltrimethoxysilane.

The use of silane coupling reagents, especially those of the formulaR′Si(OR)₃ in which R′ is typically an aliphatic group with a terminalamine and R is a lower alkyl group, to promote adhesion is known in theart (see, e.g. U.S. Pat. No. 5,212,050 which is incorporated herein byreference). For example, chemically modified electrodes in which asilane such as γ-aminopropyltriethoxysilane and glutaraldehyde were usedin a step-wise process to attach and to co-crosslink bovine serumalbumin (BSA) and glucose oxidase (GOx) to the electrode surface arewell known in the art (see, e.g. Yao, T. Analytica Chim. Acta 1983, 148,27-33).

In certain embodiments of the invention, the adhesion promotingconstituent further comprises one or more compounds that can also bepresent in an adjacent constituent such as the polydimethyl siloxane(PDMS) compounds that serves to limit the diffusion of analytes such asglucose through the analyte modulating constituent. In illustrativeembodiments the formulation comprises 0.5-20% PDMS, typically 5-15%PDMS, and most typically 10% PDMS. In certain embodiments of theinvention, the adhesion promoting constituent is crosslinked within thelayered sensor system and correspondingly includes an agent selected forits ability to crosslink a moiety present in a proximal constituent suchas the analyte modulating constituent. In illustrative embodiments ofthe invention, the adhesion promoting constituent includes an agentselected for its ability to crosslink an amine or carboxyl moiety of aprotein present in a proximal constituent such a the analyte sensingconstituent and/or the protein constituent and or a siloxane moietypresent in a compound disposed in a proximal layer such as the analytemodulating layer.

Analyte Modulating Constituent

The electrochemical sensors of the invention include an analytemodulating constituent disposed on the sensor (see, e.g. element 112 inFIG. 2). The term “analyte modulating constituent” is used hereinaccording to art accepted terminology and refers to a constituent thattypically forms a membrane on the sensor that operates to modulate thediffusion of one or more analytes, such as glucose, through theconstituent. In certain embodiments of the invention, the analytemodulating constituent is an analyte-limiting membrane which operates toprevent or restrict the diffusion of one or more analytes, such asglucose, through the constituents. In other embodiments of theinvention, the analyte-modulating constituent operates to facilitate thediffusion of one or more analytes, through the constituents. Optionallysuch analyte modulating constituents can be formed to prevent orrestrict the diffusion of one type of molecule through the constituent(e.g. glucose), while at the same time allowing or even facilitating thediffusion of other types of molecules through the constituent (e.g. O₂).

With respect to glucose sensors, in known enzyme electrodes, glucose andoxygen from blood, as well as some interferents, such as ascorbic acidand uric acid, diffuse through a primary membrane of the sensor. As theglucose, oxygen and interferents reach the analyte sensing constituent,an enzyme, such as glucose oxidase, catalyzes the conversion of glucoseto hydrogen peroxide and gluconolactone. The hydrogen peroxide maydiffuse back through the analyte modulating constituent, or it maydiffuse to an electrode where it can be reacted to form oxygen and aproton to produce a current that is proportional to the glucoseconcentration. The sensor membrane assembly serves several functions,including selectively allowing the passage of glucose therethrough. Inthis context, an illustrative analyte modulating constituent is asemi-permeable membrane which permits passage of water, oxygen and atleast one selective analyte and which has the ability to absorb water,the membrane having a water soluble, hydrophilic polymer.

A variety of illustrative analyte modulating compositions are known inthe art and are described for example in U.S. Pat. Nos. 6,319,540,5,882,494, 5,786,439 5,777,060, 5,771,868 and 5,391,250, the disclosuresof each being incorporated herein by reference. The hydrogels describedtherein are particularly useful with a variety of implantable devicesfor which it is advantageous to provide a surrounding water constituent.In some embodiments of the invention, the analyte modulating compositionincludes PDMS. In certain embodiments of the invention, the analytemodulating constituent includes an agent selected for its ability tocrosslink a siloxane moiety present in a proximal constituent. Inclosely related embodiments of the invention, the adhesion promotingconstituent includes an agent selected for its ability to crosslink anamine or carboxyl moiety of a protein present in a proximal constituent.

In some embodiments of the invention, the analyte modulating layer isformed to comprise a blended mixture of a linear polyurethane/polyureapolymer and a branched acrylate polymer such as those disclosed in U.S.patent application Ser. No. 12/643,790, the contents of which areincorporated by reference. Typically these polymers are blended togetherat a ratio of between 1:1 and 1:20 by weight %, with thepolyurethane/polyurea polymer being formed from a mixture comprising adiisocyanate; a hydrophilic polymer comprising a hydrophilic diol orhydrophilic diamine; and a siloxane having an amino, hydroxyl orcarboxylic acid functional group at a terminus; and the branchedacrylate polymer formed from a mixture comprising a butyl, propyl, ethylor methyl-acrylate; an amino-acrylate; a siloxane-acrylate; and apoly(ethylene oxide)-acrylate. Typically the analyte modulating layer isformed to exhibit a permeability to glucose that changes less than 2%per degree centigrade over a temperature range of 22 to 40 degreescentigrade.

Cover Constituent

The electrochemical sensors of the invention include one or more coverconstituents which are typically electrically insulating protectiveconstituents (see, e.g. element 106 in FIG. 2). Typically, such coverconstituents can be in the form of a coating, sheath or tube and aredisposed on at least a portion of the analyte modulating constituent.Acceptable polymer coatings for use as the insulating protective coverconstituent can include, but are not limited to, non-toxic biocompatiblepolymers such as silicone compounds, polyimides, biocompatible soldermasks, epoxy acrylate copolymers, or the like. Further, these coatingscan be photo-imageable to facilitate photolithographic forming ofapertures through to the conductive constituent. A typical coverconstituent comprises spun on silicone. As is known in the art, thisconstituent can be a commercially available RTV (room temperaturevulcanized) silicone composition. A typical chemistry in this context ispolydimethyl siloxane (acetoxy based).

C. Typical Analyte Sensor System Embodiments of the Invention

Embodiments of the sensor elements and sensors can be operativelycoupled to a variety of other systems elements typically used withanalyte sensors (e.g. structural elements such as piercing members,insertion sets and the like as well as electronic components such asprocessors, monitors, medication infusion pumps and the like), forexample to adapt them for use in various contexts (e.g. implantationwithin a mammal). One embodiment of the invention includes a method ofmonitoring a physiological characteristic of a user using an embodimentof the invention that includes an input element capable of receiving asignal from a sensor that is based on a sensed physiologicalcharacteristic value of the user, and a processor for analyzing thereceived signal. In typical embodiments of the invention, the processordetermines a dynamic behavior of the physiological characteristic valueand provides an observable indicator based upon the dynamic behavior ofthe physiological characteristic value so determined. In someembodiments, the physiological characteristic value is a measure of theconcentration of blood glucose in the user. In other embodiments, theprocess of analyzing the received signal and determining a dynamicbehavior includes repeatedly measuring the physiological characteristicvalue to obtain a series of physiological characteristic values in orderto, for example, incorporate comparative redundancies into a sensorapparatus in a manner designed to provide confirmatory information onsensor function, analyte concentration measurements, the presence ofinterferences and the like.

Embodiments of the invention include devices which display data frommeasurements of a sensed physiological characteristic (e.g. bloodglucose concentrations) in a manner and format tailored to allow a userof the device to easily monitor and, if necessary, modulate thephysiological status of that characteristic (e.g. modulation of bloodglucose concentrations via insulin administration). An illustrativeembodiment of the invention is a device comprising a sensor inputcapable of receiving a signal from a sensor, the signal being based on asensed physiological characteristic value of a user; a memory forstoring a plurality of measurements of the sensed physiologicalcharacteristic value of the user from the received signal from thesensor; and a display for presenting a text and/or graphicalrepresentation of the plurality of measurements of the sensedphysiological characteristic value (e.g. text, a line graph or the like,a bar graph or the like, a grid pattern or the like or a combinationthereof. Typically, the graphical representation displays real timemeasurements of the sensed physiological characteristic value. Suchdevices can be used in a variety of contexts, for example in combinationwith other medical apparatuses. In some embodiments of the invention,the device is used in combination with at least one other medical device(e.g. a glucose sensor).

An illustrative system embodiment consists of a glucose sensor, atransmitter and pump receiver and a glucose meter. In this system, radiosignals from the transmitter can be sent to the pump receiver every 5minutes to provide providing real-time sensor glucose (SG) values.Values/graphs are displayed on a monitor of the pump receiver so that auser can self monitor blood glucose and deliver insulin using their owninsulin pump. Typically an embodiment of device disclosed hereincommunicates with a second medical device via a wired or wirelessconnection. Wireless communication can include for example the receptionof emitted radiation signals as occurs with the transmission of signalsvia RF telemetry, infrared transmissions, optical transmission, sonicand ultrasonic transmissions and the like. Optionally, the device is anintegral part of a medication infusion pump (e.g. an insulin pump).Typically in such devices, the physiological characteristic valuesincludes a plurality of measurements of blood glucose.

FIG. 3 provides a perspective view of one generalized embodiment ofsubcutaneous sensor insertion system and a block diagram of a sensorelectronics device according to one illustrative embodiment of theinvention. Additional elements typically used with such sensor systemembodiments are disclosed for example in U.S. Patent Application No.20070163894, the contents of which are incorporated by reference. FIG. 3provides a perspective view of a telemetered characteristic monitorsystem 1, including a subcutaneous sensor set 10 provided forsubcutaneous placement of an active portion of a flexible sensor 12, orthe like, at a selected site in the body of a user. The subcutaneous orpercutaneous portion of the sensor set 10 includes a hollow, slottedinsertion needle 14 having a sharpened tip 44, and a cannula 16. Insidethe cannula 16 is a sensing portion 18 of the sensor 12 to expose one ormore sensor electrodes 20 to the user's bodily fluids through a window22 formed in the cannula 16. The sensing portion 18 is joined to aconnection portion 24 that terminates in conductive contact pads, or thelike, which are also exposed through one of the insulative layers. Theconnection portion 24 and the contact pads are generally adapted for adirect wired electrical connection to a suitable monitor 200 coupled toa display 314 for monitoring a user's condition in response to signalsderived from the sensor electrodes 20. The connection portion 24 may beconveniently connected electrically to the monitor 200 or acharacteristic monitor transmitter 400 by a connector block 28 (or thelike) as shown and described in U.S. Pat. No. 5,482,473, entitled FLEXCIRCUIT CONNECTOR, which is incorporated by reference.

As shown in FIG. 3, in accordance with embodiments of the presentinvention, subcutaneous sensor set 10 may be configured or formed towork with either a wired or a wireless characteristic monitor system.The proximal part of the sensor 12 is mounted in a mounting base 30adapted for placement onto the skin of a user. The mounting base 30 canbe a pad having an underside surface coated with a suitable pressuresensitive adhesive layer 32, with a peel-off paper strip 34 normallyprovided to cover and protect the adhesive layer 32, until the sensorset 10 is ready for use. The mounting base 30 includes upper and lowerlayers 36 and 38, with the connection portion 24 of the flexible sensor12 being sandwiched between the layers 36 and 38. The connection portion24 has a forward section joined to the active sensing portion 18 of thesensor 12, which is folded angularly to extend downwardly through a bore40 formed in the lower base layer 38. Optionally, the adhesive layer 32(or another portion of the apparatus in contact with in vivo tissue)includes an anti-inflammatory agent to reduce an inflammatory responseand/or anti-bacterial agent to reduce the chance of infection. Theinsertion needle 14 is adapted for slide-fit reception through a needleport 42 formed in the upper base layer 36 and further through the lowerbore 40 in the lower base layer 38. After insertion, the insertionneedle 14 is withdrawn to leave the cannula 16 with the sensing portion18 and the sensor electrodes 20 in place at the selected insertion site.In this embodiment, the telemetered characteristic monitor transmitter400 is coupled to a sensor set 10 by a cable 202 through a connector 204that is electrically coupled to the connector block 28 of the connectorportion 24 of the sensor set 10.

In the embodiment shown in FIG. 3, the telemetered characteristicmonitor 400 includes a housing 206 that supports a printed circuit board208, batteries 210, antenna 212, and the cable 202 with the connector204. In some embodiments, the housing 206 is formed from an upper case214 and a lower case 216 that are sealed with an ultrasonic weld to forma waterproof (or resistant) seal to permit cleaning by immersion (orswabbing) with water, cleaners, alcohol or the like. In someembodiments, the upper and lower case 214 and 216 are formed from amedical grade plastic. However, in alternative embodiments, the uppercase 214 and lower case 216 may be connected together by other methods,such as snap fits, sealing rings, RTV (silicone sealant) and bondedtogether, or the like, or formed from other materials, such as metal,composites, ceramics, or the like. In other embodiments, the separatecase can be eliminated and the assembly is simply potted in epoxy orother moldable materials that is compatible with the electronics andreasonably moisture resistant. As shown, the lower case 216 may have anunderside surface coated with a suitable pressure sensitive adhesivelayer 118, with a peel-off paper strip 120 normally provided to coverand protect the adhesive layer 118, until the sensor set telemeteredcharacteristic monitor transmitter 400 is ready for use.

In the illustrative embodiment shown in FIG. 3, the subcutaneous sensorset 10 facilitates accurate placement of a flexible thin filmelectrochemical sensor 12 of the type used for monitoring specific bloodparameters representative of a user's condition. The sensor 12 monitorsglucose levels in the body, and may be used in conjunction withautomated or semi-automated medication infusion pumps of the external orimplantable type as described in U.S. Pat. No. 4,562,751; 4,678,408;4,685,903 or 4,573,994, to control delivery of insulin to a diabeticpatient.

In the illustrative embodiment shown in FIG. 3, the sensor electrodes 10may be used in a variety of sensing applications and may be configuredin a variety of ways. For example, the sensor electrodes 10 may be usedin physiological parameter sensing applications in which some type ofbiomolecule is used as a catalytic agent. For example, the sensorelectrodes 10 may be used in a glucose and oxygen sensor having aglucose oxidase enzyme catalyzing a reaction with the sensor electrodes20. The sensor electrodes 10, along with a biomolecule or some othercatalytic agent, may be placed in a human body in a vascular ornon-vascular environment. For example, the sensor electrodes 20 andbiomolecule may be placed in a vein and be subjected to a blood stream,or may be placed in a subcutaneous or peritoneal region of the humanbody.

In the embodiment of the invention shown in FIG. 3, the monitor ofsensor signals 200 may also be referred to as a sensor electronicsdevice 200. The monitor 200 may include a power source, a sensorinterface, processing electronics (i.e. a processor), and dataformatting electronics. The monitor 200 may be coupled to the sensor set10 by a cable 202 through a connector that is electrically coupled tothe connector block 28 of the connection portion 24. In an alternativeembodiment, the cable may be omitted. In this embodiment of theinvention, the monitor 200 may include an appropriate connector fordirect connection to the connection portion 204 of the sensor set 10.The sensor set 10 may be modified to have the connector portion 204positioned at a different location, e.g., on top of the sensor set tofacilitate placement of the monitor 200 over the sensor set.

D. Embodiments of the Invention and Associated Characteristics

Embodiments of the invention disclosed herein focus on implantableanalyte sensors and sensor systems that are designed to include elementsand/or configurations of elements that facilitate sensor initializationand/or start-up in vivo (e.g. the run-in time that it takes for a sensorto settle into its environment and start transmitting meaningfulinformation after being implanted in vivo). In particular, it is knownin the art that the amount time required for sensor initializationand/or start-up prior to its use can be relatively long (e.g. inamperometric glucose sensors, the sensor start-up initialization timescan range from 2 to 10 hours), a factor which can hinder the use of suchsensors in the administration of medical care. For example, in hospitalsettings, a relatively long sensor initialization and/or start-up periodcan delay the receipt of important information relating to patienthealth (e.g. hyperglycemia or hypoglycemia in a diabetic patient),thereby delaying treatments predicated on the receipt of suchinformation (e.g. the administration of insulin). In addition, arelatively long sensor initialization and/or start-up period in hospitalsettings can require repeated monitoring by hospital staff, a factorwhich contributes to the costs of patient care. For these reasons,sensors having reduced initialization and/or start-up times in vivo inhospital settings and sensors and sensor systems that are designed toinclude elements and/or configurations of elements that diminish longsensor initialization and/or start-up times are highly desirable. Withglucose sensors for example, a 15-30 minute reduction of sensorinitialization and/or start-up time is highly desirable because, forexample, such shorter initialization times can: (1) reduce the need forpatient monitoring by hospital personnel, a factor which contributes tothe cost-effectiveness of such medical devices; and (2) reduce delays inthe receipt of important information relating to patient health.

In individuals using analyte sensors in non-hospital settings (e.g.diabetics using glucose sensors to manage their disease), relativelylong sensor initialization and/or start-up periods are alsoproblematical due to both the inconvenience to the user as well as thedelayed receipt of information relating to user health. The use ofglucose sensors, insulin infusion pumps and the like in the managementof diabetes has increased in recent years due for example to studiesshowing that the morbidity and mortality issues associated with thischronic disease decrease dramatically when a patient administers insulinin a manner that closely matches the rise and fall of physiologicalinsulin concentrations in healthy individuals. Consequently, patientswho suffer from chronic diseases such as diabetes are instructed bymedical personnel to play an active role in the management of theirdisease, in particular, the close monitoring and modulation of bloodglucose levels. In this context, because many diabetics do not havemedical training, they may forgo optimal monitoring and modulation ofblood glucose levels due to complexities associated with suchmanagement, for example, a two hour start-up period which can be aninconvenience in view of a patient's active daily routine. For thesereasons, sensors and sensor systems that are designed to includeelements and/or configurations of elements can reduce sensorinitialization and/or start-up times in are highly desirable insituations where such sensors are operated by a diabetic patient withoutmedical training because they facilitate the patient's convenientmanagement of their disease, behavior which is shown to decrease thewell known morbidity and mortality issues observed in individualssuffering from chronic diabetes.

While the analyte sensor and sensor systems disclosed herein aretypically designed to be implantable within the body of a mammal, theinventions disclosed herein are not limited to any particularenvironment and can instead be used in a wide variety of contexts, forexample for the analysis of most in vivo and in vitro liquid samplesincluding biological fluids such as interstitial fluids, whole-blood,lymph, plasma, serum, saliva, urine, stool, perspiration, mucus, tears,cerebrospinal fluid, nasal secretion, cervical or vaginal secretion,semen, pleural fluid, amniotic fluid, peritoneal fluid, middle earfluid, joint fluid, gastric aspirate or the like. In addition, solid ordesiccated samples may be dissolved in an appropriate solvent to providea liquid mixture suitable for analysis.

The invention disclosed herein has a number of embodiments. Oneillustrative embodiment of the invention is an analyte sensor apparatuscomprising: an elongated (i.e. having notably more length than width)base layer; a conductive layer disposed on the base layer and comprisinga reference electrode, a working electrode and a counter electrode; ananalyte sensing layer disposed on the conductive layer; an analytemodulating layer disposed on the analyte sensing layer, wherein theanalyte modulating layer comprises a composition that modulates thediffusion of an analyte diffusing through the analyte modulating layer;and a cover layer disposed on the analyte sensor apparatus, wherein thecover layer comprises an aperture positioned on the cover layer so as tofacilitate an analyte contacting and diffusing through the analytemodulating layer and contacting the analyte sensing layer. Typicalembodiments of the invention are comprised of biocompatible materialsand/or have structural features designed for implantation within amammal. Methodological embodiments of the invention include methods formaking and using the sensor embodiments disclosed herein. Certainembodiments of the invention include methods of using a specific sensorelement and/or a specific constellation of sensor elements to produceand/or facilitate one or more functions of the sensor embodimentsdisclosed herein.

As disclosed herein, those of skill in the art understand that aconductive layer disposed on the base layer and comprising a workingelectrode, a counter electrode and a reference electrode includesembodiments wherein the conductive layer is disposed on at least aportion the base layer and does not necessarily completely cover thebase layer. Those of skill in the art will understand that this refersto other layers within the sensor, with for example, an analyte sensinglayer disposed on the conductive layer encompassing sensor embodimentswhere the analyte sensing layer disposed on at least a portion of theconductive layer; and an analyte modulating layer disposed on theanalyte sensing encompassing an analyte modulating layer disposed on atleast a portion of the analyte sensing etc. etc. Optionally, theelectrodes can be disposed on a single surface or side of the sensorstructure. Alternatively, the electrodes can be disposed on a multiplesurfaces or sides of the sensor structure (and can for example beconnected by vias through the sensor material(s) to the surfaces onwhich the electrodes are disposed). In certain embodiments of theinvention, the reactive surfaces of the electrodes are of differentrelative areas/sizes, for example a 1× reference electrode, a 2.6×working electrode and a 3.6× counter electrode.

In certain embodiments of the invention, an element of the apparatussuch as an electrode or an aperture is designed to have a specificconfiguration and/or is made from a specific material and/or ispositioned relative to the other elements so as to facilitate a functionof the sensor. For example, without being bound by a specific theory ormechanism of action, it appears that sensor embodiments (e.g. simplethree electrode embodiments) may be more susceptible to localenvironment changes around a single electrode. For example, a gas bubbleon top of or close to a reference or another electrode, and/or astagnating or semi-stagnating pool of fluid on top of or close to areference or another electrode may consequently compromises sensorperformance. In this context, a distributed electrode configurationappears be advantageous because the distribution of the electrode areaallows the sensor to compensate for signal lost to a small local area(e.g. as can occur due to lack of hydration, fluid stagnation, apatient's immune response, or the like).

In some sensor embodiments, the distributed electrodes areorganized/disposed within a flex-circuit assembly (i.e. a circuitryassembly that utilizes flexible rather than rigid materials). Suchflex-circuit assembly embodiments provide an interconnected assembly ofelements (e.g. electrodes, electrical conduits, contact pads and thelike) configured to facilitate wearer comfort (for example by reducingpad stiffness and wearer discomfort) as well as parameter measurementperformance and are disclosed in more detail in U.S. patent applicationSer. Nos. 12/184,046 (filed Jul. 31, 2008), which is hereby incorporatedby reference.

Typical analyte sensor apparatus embodiments comprises a plurality ofworking electrodes, counter electrodes and reference electrodes.Optionally, the plurality of working, counter and reference electrodesare grouped together as a unit and positionally distributed on theconductive layer in a repeating pattern of units. Alternatively, theplurality of working, counter and reference electrodes are groupedtogether and positionally distributed on the conductive layer in anon-repeating pattern of units. In certain embodiments of the invention,the elongated base layer is made from a material that allows the sensorto twist and bend when implanted in vivo; and the electrodes are groupedin a configuration that facilitates an in vivo fluid contacting at leastone of working electrode as the sensor apparatus twists and bends whenimplanted in vivo. In some embodiments, the electrodes are grouped in aconfiguration that allows the sensor to continue to maintain an optimalfunction if a portion of the sensor having one or more electrodes isdislodged from an in vivo environment and exposed to an ex vivoenvironment.

Typically, the electrodes in a sensor are of a rectangular shape, i.e.have a longer side and a shorter side (including those of a rectangularshape, yet having rounded edges). In some embodiments of the invention,the electrode configuration is such that a longer side of at least oneof the electrodes in a distributed electrode pattern is parallel to anlonger side of at least one of the other electrodes in the distributedelectrode pattern (and optionally all of the electrodes in thedistributed electrode pattern). As disclosed in U.S. patent applicationSer. Nos. 12/184,046 (filed Jul. 31, 2008), incorporated herein byreference, sensor embodiments having such configurations are observed toexhibit a better start-up profile than sensors without electrodesconfigured in this pattern. In some embodiments of the invention, anaperture is positioned on the cover layer so that a fluid comprising theanalyte contacts the reference electrode, the working electrode and thecounter electrode in a sequential manner so as to facilitate sensorhydration and/or sensor start-up or initialization.

Various elements of the sensor apparatus can be disposed at a certainlocation in the apparatus and/or configured in a certain shape and/or beconstructed from a specific material so as to facilitate strength,hydration and/or function of the sensor. One such embodiment of theinvention includes an elongated base comprised of a polyimmide ordielectric ceramic material that facilitates the strength and durabilityof the sensor. In certain embodiments of the invention, the structuralfeatures and/or relative position of the working and/or counter and/orreference electrodes is designed to influence sensor manufacture, useand/or function. One such embodiment of the invention includeselectrodes having one or more rounded edges so as to inhibitdelamination of a layer disposed on the electrode (e.g. an analytesensing layer comprising glucose oxidase). Related embodiments of theinvention include methods for inhibiting delamination of a sensor layerusing a sensor embodiments of the invention (e.g. one having one or moreelectrodes having one or more rounded edges).

In some embodiments of the invention, a barrier element is disposed onthe apparatus so as to inhibit spreading of a layer disposed on anelectrode. Optionally, an element such as a metallic or other structureis disposed on top of the dam structure(s). Related embodiments of theinvention include methods for inhibiting movement of a compound disposedon a sensor embodiments of the invention (e.g. one constructed to havesuch a barrier structure). Optionally, a barrier element is disposed onthe apparatus so as to encircle a reactive surface of an electrode. Suchbarrier elements can be made from a variety of materials, for example apolyimmide. In various embodiments of the invention, these elements canbe formed as part of the electrode or alternatively bonded to theelectrode after it is formed (e.g. using an epoxy or the like).

In some embodiments of the invention, at least one electrode is formedfrom a flexible electrically conductive wire. Optionally, the flexibleelectrically conductive wire is disposed in the apparatus in a coiledconfiguration. In certain embodiments, the wire electrode is formed froma platinum, a silver and/or a palladium composition. Optionally, thewire electrode is disposed within a tube cover having at least 5, 10 or15 apertures positioned so that an analyte of interest can contact thewire electrode. Embodiments of the invention that comprise a wireelectrode and/or a distributed electrode pattern such as those disclosedabove can be used in methods designed to diminish or overcome problemsassociated with the shaking and bumping of potentially fragileelectronic elements that occurs when an apparatus flexes as it is usedin vivo. In particular, an apparatus implanted in vivo is subjected to avariety of mechanical stresses during a patient's daily routine ofactivities (e.g. stretching, bending, walking and the like). Suchstresses are known in the art to have the ability to damage elementswithin a device, in particular electrodes, which can be brittle andprone to breakage. Embodiments of the invention are designed to overcomeproblems by using elements (e.g. a flexible wire electrode) and/orarchitectures (e.g. distributed electrode configurations) that are lesslikely to lose optimal function as a result of the mechanical stressesthat result from a patient's daily routine of activities.

Another embodiment of the invention is a multiconductor sensorcomprising a series of electrodes disposed on a base such as a ribboncable. This configuration is useful in manufacturing/production of thesensor, for example those processes that involve progressive laserablation. In one such embodiment, a pattern of laser ablation iscontrolled to produce a single wire with one or more working, counterand reference electrodes and/or a plurality of such electrode groups.Optionally this is in a reel form that is cut into segments prior tosensor manufacture. One illustrative embodiment of this design comprisesa wire electrode with multiple reading points (e.g. perforations) alongits wire/ribbon body. This wire can further be disposed within sheath ortube having a plurality of windows. Subsequent layers such as theanalyte modulating layer can be coated over a portion of, oralternatively, the whole wire. Related embodiments of the inventioninclude a method of making such sensors, wherein a step in the methodincludes disposing the wire electrode in the form of a reel that is thencut into segments during the manufacturing process.

In certain embodiments of the invention, an electrode of the apparatuscomprises a platinum composition and the apparatus further comprises atitanium composition disposed between the elongated base layer and theconductive layer. Optionally in such embodiments, apparatus furthercomprises a gold composition disposed between the titanium compositionand the conductive layer. Certain embodiments form one or more of theselayers via a process that includes photolithography. Such embodiments ofthe invention typically exhibit enhanced bonding between layeredmaterials within the sensor and/or less corrosion and/or improvedbiocompatibility profiles. Such materials are used for example to makesensors having a reduced corrosion profile, one that allows certaincorrosion inhibiting insulating elements to be eliminated from a sensordesign. Related embodiments of the invention include methods forinhibiting corrosion of a sensor element and/or method for improving thebiocompatibility of a sensor embodiments of the invention (e.g. oneconstructed to use such materials). For certain methods that can be usedto make such embodiments of the invention, see, e.g. U.S. Pat. No.7,033,336, the contents of which are incorporated by reference.

In addition, electrodes in various embodiments of the invention can becoated with a variety of materials (e.g. an analyte modulating layer) inorder to influence the function of the sensor apparatus. In someembodiments of the invention, a hydrophilic analyte modulating layer iscoated over at least 50, 75% or 100% of the reactive surface of anelectrode (e.g. an electrically conductive wire). For example certainembodiments of the invention disclosed herein (e.g. amperometric glucosesensors) include elements and/or constellations of elements that aredesigned to overcome what is known as “oxygen deficit problem.” Thisproblem relates to the fact in that sensors designed to measure ananalyte via the reaction of an analyte and oxygen, the oxygenconcentration must be in excess. If the oxygen is not in excess (and isinstead the rate limiting reactant), the sensor signal will beproportional to the oxygen concentration and not the analyte which thesensor is designed to measure. Under these conditions, sensors will notfunction properly. Therefore, there is a need for sensors that includebiocompatible membrane with differential oxygen and analytepermeabilities (e.g. glucose limiting membranes) and further havingelements that function to enhance sensor initialization start up timeand further.

Optionally, embodiments of the invention include a plurality of workingelectrodes and/or counter electrodes and/or reference electrodes (e.g.to provide redundant sensing capabilities). Such embodiments of theinvention can be used in embodiments of the invention that include aprocessor (e.g. one linked to a program adapted for a signalsubtraction/cancellation process) are designed factor out backgroundsignals in vivo, for example by comparing signal(s) at GOx coatedworking electrode with signal at working electrode not coated with GOx(e.g. background detection followed by a signal subtraction/cancellationprocess to arrive at a true signal). Certain of these embodiments of theinvention are particularly useful for sensing glucose at the upper andlower ends of the glucose signal curves. Similar embodiments of theinvention are used to factor out interference, for example by comparingsignal(s) at GOx coated working electrode with signal at workingelectrode not coated with GOx. Embodiments of the invention can includea coating of a Prussian blue composition on an electrode at a locationand in an amount sufficient to mediate an electrical potential of anelectrode of the apparatus. Related embodiments of the invention includemethods for mediating an electrical potential of an electrode of thedisclosed sensor apparatus (e.g. by using a Prussian blue composition).Prussian Blue formulas are known in the art and includeFe4[Fe(CN6]3×H₂O, CI no. 77510 and Ke[Fe(Cn)6]×H₂O id CI no. 77520.

In some embodiments of the invention, the architecture or thickness of asensor layer is used to optimize a property of the sensor. For examplein some embodiments of the invention, the elongated base layer iscomprised of a dielectric or polyimmide ceramic material that is atleast 100 microns thick. In some embodiments of the invention, theanalyte modulating layer is at least 6, 7, 8, 9, 10, 15, 20, 25 or 30microns thick. Certain embodiments of the invention use a thick layer(e.g. 25 or 30 microns) of an analyte modulating layer because in suchembodiments, this thick layer is observed to both optimize the linearityof an analyte signal over a range of signals (e.g. glucoseconcentration). Such thick layers have further properties that aredesirable in certain embodiments of the invention, for example a longeranalyte modulating layer lifetime (e.g. due to the extra material), aproperty that makes them particularly suited for certain long termsensor embodiments.

Typical embodiments of the invention comprise further layers such as anadhesion promoting layer disposed between the analyte sensing layer andthe analyte modulating layer. Optionally in such embodiments, a firstcompound in the adhesion promoting layer is crosslinked to a secondcompound in the analyte sensing layer. Certain embodiments of theinvention include an interference rejection layer, for example onecomprised of a NAFION (a sulfonated tetrafluorethylene copolymer havingthe molecular formula C7HF13O5S. C2F4, CAS number [31175-20-9]) and/or acellulose acetate composition. An interference rejection membrane (IRM)may comprise NAFION and its effectiveness at inhibiting interferingsignals that can be generated by acetominophenol in an amperometricsensor. Typically, an IRM is disposed under an analyte sensing layer(e.g. one comprising glucose oxidase). In certain embodiments of theinvention, the IRM is disposed between the reactive surface of anelectrode and an analyte sensing layer. Related embodiments of theinvention include methods for inhibiting one or more signals generatedby an interfering compound in various sensor embodiments of theinvention (e.g. by using an interference rejection layer).

In typical embodiments of the invention, the sensor is operativelycoupled to further elements (e.g. electronic components) such aselements designed to transmit and/or receive a signal, monitors, pumps,processors and the like. For example, in some embodiments of theinvention, the sensor is operatively coupled to a sensor input capableof receiving a signal from the sensor that is based on a sensedphysiological characteristic value in the mammal; and a processorcoupled to the sensor input, wherein the processor is capable ofcharacterizing one or more signals received from the sensor. A widevariety of sensor configurations as disclosed herein can be used in suchsystems. Optionally, for example, the sensor comprises three workingelectrodes, one counter electrode and one reference electrode. Incertain embodiments, at least one working electrode is coated with ananalyte sensing layer comprising glucose oxidase (and optionally two arecoated with GOx) and at least one working electrode is not coated withan analyte sensing layer comprising glucose oxidase. Such embodiments ofthe invention can be used for example in sensor embodiments designedfactor out background signals in vivo, for example by comparingsignal(s) at GOx coated working electrode(s) with signal at workingelectrode(s) not coated with GOx (e.g. background detection followed bya signal subtraction/cancellation process to arrive at a true signal).

Embodiments of the invention include sensors and sensor systems havingconfigurations of elements and/or architectures that optimize aspects ofsensor function. For example, certain embodiments of the invention areconstructed to include multiple and/or redundant elements such asmultiple sets of sensors and/or sensor system elements such as multiplepiercing members (e.g. needles) and/or a cannulas organized on aninsertion apparatus for use at a patient's in vivo insertion site. Forexample, sensor sets may include dual piercing members as disclosed inU.S. patent application Ser. No. 13/008,723, filed Jan. 18, 2011, whichis herein incorporated by reference. This embodiment of the invention isa sensor apparatus for monitoring a body characteristic of the patient,the apparatus comprising a base element adapted to secure the apparatusto the patient, a first piercing member that is coupled to and extendingfrom the base element, wherein the first piercing member is operativelycoupled to (e.g. to provide structural support and/or enclose) at leastone first electrochemical sensor having at least one electrode fordetermining at least one body characteristic of the patient at a firstsensor placement site, as well as a second piercing member that iscoupled to and extending from the base element and operatively coupledto at least one second electrochemical sensor having at least oneelectrode for determining at least one body characteristic of thepatient at a second sensor placement site. In some embodiments of theinvention, such sensor systems are used in a hospital setting such as inan intensive care unit (e.g. to measure blood glucose concentrations inthe interstitial fluid or blood of a diabetic patient). In otherembodiments of the invention, the apparatus is used in an ambulatorycontext, for example by a diabetic in the daily monitoring of bloodglucose.

Embodiments of the sensor systems disclosed herein are typically coupledto elements that facilitate their use in vivo such as flexible sensorsubstrates (e.g. to facilitate their use in an ambulatory context). Insuch embodiments, one or more sensing sets can be coupled to each flexsubstrate; for example an embodiment having four sensing sets, twocoupled to each substrate. A wide variety of materials known in the artcan be used to make such embodiments of the invention. In suchembodiments, a cable can carry a current (e.g. a low current such as 10nA) analogue signal with minimal noise over some distance (e.g. up toabout 3, 4, 5, 6, 7, 8, 9 or 10 feet in length). The wires used to carrythis current can be shielded in order to minimize noise due tointerference/cross-talk, for example by using either a metal mesh (e.g.a braided and/or woven mesh), a solid metal wrap (e.g. one akin toaluminum foil), or are wound individual metal wires (e.g. akin towrapping many hair like wires around to an assembly achieve the sameeffect as a solid wrap). The connector can be made of a material such asa rigid plastic (polycarbonate, ABS, polyethylene, PVC, vinyl, etc.).The outer sleeve of the cable can be made of a flexible/cleanable(resistant to chemicals) material such as polyurethane, vinyl, silicone,polyamide, etc. The material on the body can be a flexible base, forexample one similar to that used in pediatric pulse-ox sensors or clothBand-Aids.

As noted above, in certain sensor system embodiments, the electrodes areorganized/disposed within a flex-circuit assembly. In such embodimentsof the invention, the architecture of the sensor system including theflex substrates/sensors are separated by a distance great enough that:(1) a first sensor does not influence a signal etc. generated by asecond sensor (and vice versa); and (2) they sense from separate tissueenvelopes; so the signals from separate sensors do not interact. At thesame time, in typical embodiments of the invention the flex substrateswill be close enough so that they are easily packaged together and havea single insertion action.

One embodiment of the invention is an apparatus for monitoring ananalyte in a patient, the apparatus comprising: a base element adaptedto secure the apparatus to the patient; a first piercing member coupledto and extending from the base element; a first electrochemical sensoroperatively coupled to the first piercing member and comprising a firstelectrochemical sensor electrode for determining at least onephysiological characteristic of the patient at a first electrochemicalsensor placement site; a second piercing member coupled to and extendingfrom the base element; a second electrochemical sensor operativelycoupled to the second piercing member and comprising a secondelectrochemical sensor electrode for determining at least onephysiological characteristic of the patient at a second electrochemicalsensor placement site. In such embodiments of the invention, at leastone physiological characteristic monitored by the first or the secondelectrochemical sensor comprises a concentration of a naturallyoccurring analyte in the patient; the first piercing member disposes thefirst electrochemical sensor in a first tissue compartment of thepatient and the second piercing member disposes the secondelectrochemical sensor in a second tissue compartment of the patient;and the first and second piercing members are disposed on the base in aconfiguration selected to avoid a physiological response that can resultfrom implantation of the first electrochemical sensor from altering asensor signal generated by the second electrochemical sensor.

In some embodiments of the invention, the base comprises a flexiblesensor substrate (e.g. one made from a polymeric composition such as apolyimmide) that twists and bends in response to movement of implantedelectrochemical sensors in the patient. Various sensor elements such ascontact pads are typically disposed on such flexible sensor substrates.In some embodiments of the invention, the contact pads can be arrangednear the edges of the flexible sensor substrate, with leads on thesubstrate connecting the sensors to the contact pads, for example toprevent the contact pads from being contaminated with the materialsbeing tested. Embodiment can include printed circuit boards having aplurality of board contact pads arranged in the same configuration asthe sensor contact pads in the sensor array. Connectors, such asconducting elastomers, stick probes, cantilever probes, conductingadhesives, wafer-to-board bonding techniques, or other contact devices,can couple the sensors with the printed circuit board by creatingcontacts between the sensor contact pads and the board contact pads,preferably the contacts are reversible and non-permanent.

Typically, the flexible sensor substrate includes a contact pad; anelectrical conduit which connects an electrochemical sensor electrode toan electrical connection element that is operatively coupled to thecontact pad. Optionally the apparatus comprises two contact padsconnected to two electrical conduits and two sets of electrochemicalsensor electrodes comprising a working, a counter and a referenceelectrode that are disposed on the flexible sensor substrate in aconfiguration such that the two contact pads are disposed together in acentral region of the flexible sensor substrate so as to facilitateelectrical connection to a power source and each of the two sets ofelectrochemical sensor electrodes is disposed on an opposite side of theflexible sensor substrate so as to maximize sensor separation, the twocontact pads are disposed together one side of the flexible sensorsubstrate so as to facilitate electrical connection to a power sourceand the two sets of electrochemical sensor electrodes are disposedtogether in a row on an opposite side of the flexible sensor substrateso as to provide a compact design for patient use; or the two contactpads are disposed together one side of the flexible sensor substrate soas to facilitate electrical connection to a power source and the twosets of electrochemical sensor electrodes are disposed together in astaggered arrangement on an opposite side of the flexible sensorsubstrate so as to provide a compact design for patient use whileproviding a greater distance between each set of electrochemical sensorelectrodes as compared to sensor electrodes not disposed together in astaggered arrangement. In typical embodiments of the invention, acontact pad and an electrode are at least, 15, 16, 17, 18, 19, 20, 21,22, 23, 24 or 25 millimeters apart.

In certain embodiments of the invention, at least one electrochemicalsensor electrode comprises oxidoreductase that generates hydrogenperoxide upon exposure to a ligand for the oxidoreductase. In someembodiments for example, the first and second electrochemical sensorcomprises a working electrode, a counter electrode and a referenceelectrode; a working electrode in the first electrochemical sensor iscoated with glucose oxidase; and no working electrode in the secondelectrochemical sensor is coated with glucose oxidase. Optionally, thefirst and second electrochemical sensors comprise a plurality ofworking, counter and reference electrodes that are grouped together as aunit and positionally distributed in a repeating pattern of units.

In some embodiments of the invention, the first and secondelectrochemical sensors are operatively coupled to a sensor inputcapable of receiving signals from the first and second electrochemicalsensors; and a processor coupled to the sensor input, wherein theprocessor is capable of characterizing one or more signals received fromthe first and second electrochemical sensors. Optionally, a pulsedvoltage is used to obtain a signal from an electrode. In certainembodiments of the invention, the processor is capable of comparing afirst signal received from a working electrode in response to a firstworking potential with a second signal received from a working electrodein response to a second working potential.

A related embodiment of the invention is an apparatus for monitoringglucose concentrations in a patient, the apparatus comprising a flexiblesensor substrate adapted to secure the apparatus to the patient; a firstpiercing member coupled to and extending from the flexible sensorsubstrate, wherein the first piercing member is operatively coupled to afirst electrochemical sensor comprising a first electrochemical sensorelectrode for measuring glucose concentrations at a firstelectrochemical sensor placement site; a second piercing member coupledto and extending from the flexible sensor substrate, wherein the secondpiercing member is operatively coupled to a second electrochemicalsensor comprising a second electrochemical sensor electrode fordetermining at least one physiological characteristic of the patient ata second electrochemical sensor placement site; a processor capable ofcharacterizing a plurality of signals received from the first and secondelectrochemical sensors. In such embodiments of the invention, the firstpiercing member disposes the first electrochemical sensor in a firsttissue compartment of the patient and the second piercing memberdisposes the second electrochemical sensor in a second tissuecompartment of the patient; and the first and second piercing membersare disposed on the flexible sensor substrate in a configurationselected to avoid a physiological response to the implantation of thefirst electrochemical sensor from effecting a sensor signal generated bythe second electrochemical sensor.

Optionally, the first and the second electrochemical sensor comprise anelongated base layer; a conductive layer disposed on the base layer andcomprising a reference electrode, a working electrode and a counterelectrode; an analyte sensing layer disposed on the conductive layer; ananalyte modulating layer disposed on the analyte sensing layer, whereinthe analyte modulating layer comprises a composition that modulates thediffusion of an analyte diffusing through the analyte modulating layer;and a cover layer disposed on the analyte sensor, wherein the coverlayer comprises an aperture positioned on the cover layer so as tofacilitate an analyte present in the patient contacting and diffusingthrough the analyte modulating layer and contacting the analyte sensinglayer. Typically, the first and second electrochemical sensors comprisea plurality of working, counter and reference electrodes that aregrouped together as a unit and positionally distributed on theconductive layer in a repeating pattern of units.

In certain embodiments of the invention, the first electrochemicalsensor comprises a working electrode coated with glucose oxidase and thesecond electrochemical sensor comprises no working electrode coated withglucose oxidase and the processor is capable of obtaining information onglucose concentrations in the patient by comparing the signals receivedfrom said working electrodes so as to identify a background signal thatis not based on a glucose concentrations in the patient. Optionally forexample, the working electrode in the first and second electrochemicalsensors are coated with glucose oxidase and the processor is capable ofobtaining information on glucose concentrations in the patient bycomparing the signals received from said working electrodes coated withglucose oxidase. In certain embodiments of the invention, the processoris used to assess sensor information derived from pulsed voltage schemethat is used to obtain a signal from an electrode. Optionally, theprocessor is capable of comparing a first signal received from a workingelectrode in response to a first working potential (e.g. 280, 535 or 635millivolts) with a second signal received from a working electrode inresponse to a second working potential (e.g. 280, 535 or 635millivolts).

In certain embodiments of the invention, the first and secondelectrochemical sensors comprise three working electrodes, one counterelectrode and one reference electrode; at least one working electrode inthe first and second electrochemical sensors is coated with glucoseoxidase; and at least one working electrode in the first and secondelectrochemical sensors is not coated with glucose oxidase. Optionally,at least two working electrodes in the first and second electrochemicalsensors are coated with glucose oxidase. Another embodiment of theinvention is a method of monitoring an analyte within the body of apatient, the method comprising implanting an analyte sensor in to thepatient, the analyte sensor comprising a base element adapted to securethe apparatus to the patient; a first piercing member coupled to andextending from the base element; a first electrochemical sensoroperatively coupled to the first piercing member and comprising a firstelectrochemical sensor electrode for determining at least onephysiological characteristic of the patient at a first electrochemicalsensor placement site; a second piercing member coupled to and extendingfrom the base element; a second electrochemical sensor operativelycoupled to the second piercing member and comprising a secondelectrochemical sensor electrode for determining at least onephysiological characteristic of the patient at a second electrochemicalsensor placement site; a sensor input capable of receiving signals fromthe first and second electrochemical sensors; and a processor coupled tothe sensor input, wherein the processor is capable of characterizing oneor more signals received from the first and second electrochemicalsensors. In such embodiments of the invention, at least onephysiological characteristic monitored by the first or the secondelectrochemical sensor comprises a concentration of a naturallyoccurring analyte in the patient; the first piercing member disposes thefirst electrochemical sensor in a first tissue compartment of thepatient and the second piercing member disposes the secondelectrochemical sensor in a second tissue compartment of the patient;the first and second piercing members are disposed on the base in aconfiguration selected to avoid a physiological response that resultsfrom implantation of the first electrochemical sensor from altering asensor signal generated by the second electrochemical sensor; andsensing alterations in current at the first and second electrochemicalsensor electrodes and correlating said alterations in current with thepresence or absence of the analyte, so that the analyte is monitored.

In some methodological embodiments of the invention, the firstelectrochemical sensor comprises a working electrode coated with glucoseoxidase and the second electrochemical sensor comprises no workingelectrode coated with glucose oxidase, the method further comprisingusing the processor to obtain information on glucose concentrations inthe patient by comparing the signals received from the workingelectrodes in said first and second electrochemical sensors so that abackground signal that is not based on a glucose concentrations in thepatient is characterized. Optionally, the first electrochemical sensorcomprises a working electrode coated with glucose oxidase and the secondelectrochemical sensor comprises a working electrode coated with glucoseoxidase, the method further comprising using the processor to obtaininformation on glucose concentrations in the patient by comparing thesignals received from the working electrodes in said first and secondelectrochemical sensors. Typically, at least one of the first and secondelectrochemical sensors is implanted in an interstitial space.

In some embodiments of sensors insertion set apparatuses, a first and asecond (and/or third etc.) electrochemical sensor comprises one working,counter and reference electrode. Alternatively, the plurality ofelectrochemical sensors comprise a plurality of working, counter andreference electrodes, for example those having a distributedconfiguration as disclosed herein. In certain embodiments of theinvention, at least two in the plurality of sensors are designed tomeasure a signal generated by the same physiological characteristic, forexample blood glucose concentration. Embodiments of the invention caninclude for example a plurality of electrochemical sensors having aworking electrode coated with an oxidoreductase such as glucose oxidaseand are used in methods designed to sample and compare glucoseconcentrations observed at the plurality of in vivo insertion sites.Alternatively, at least two in the plurality of sensors in the sensorapparatus are designed to measure signals generated by the differentcharacteristics, for example a first characteristic comprising abackground or interfering signal that is unrelated to blood glucose(e.g. “noise”) and a second characteristic comprising blood glucoseconcentrations. In an illustrative embodiment of this invention, a firstsensor is designed to measure glucose oxidase and comprises one or moreworking electrodes coated with glucose oxidase while a secondcomparative sensor is designed to measure a background or interferingsignal that is unrelated to blood glucose has no working electrode (orelectrodes) coated with glucose oxidase.

Embodiments of the invention can include a plurality of sensors coupledto a single piercing member in a manner that allows them to be disposedtogether in vivo at a single insertion site. One such embodiment of theinvention comprises a sensor apparatus for monitoring a bodycharacteristic of the patient, the apparatus comprising a base elementadapted to secure the apparatus to the patient, a first piercing membercoupled to and extending from the base element, wherein the firstpiercing member is operatively coupled to (e.g. provide structuralsupport and/or enclose) at least two electrochemical sensors having atleast one electrode for determining at least one body characteristic ofthe patient at a first sensor placement site, a second piercing membercoupled to and extending from the base element and also operativelycoupled to at least two electrochemical sensors having at least oneelectrode for determining at least one body characteristic of thepatient at a second sensor placement site. In some embodiments of theinvention, the at least two electrochemical sensors that are coupled toa piercing member are designed to measure a signal generated by the samecharacteristic, for example blood glucose concentration. Alternatively,the at least two electrochemical sensors that are coupled to a piercingmember are designed to measure signals generated by the differentcharacteristics, for example a first characteristic comprising abackground or interfering signal that is unrelated to blood glucose(e.g. “noise”) and a second characteristic comprising blood glucoseconcentrations.

Typical embodiments of the invention include a processor which comparesthe two (or more) signals produced by the plurality of sensors and, forexample, then provides a physiological characteristic reading that isbased upon the comparison of the plurality of signals. In oneillustrative embodiment, the processor uses an algorithm to providecomputational comparisons between a plurality of sensors having elementscoated with an oxidoreductase such as glucose oxidase in order to, forexample provide a comparative assessment of a physiologicalcharacteristic such as blood glucose at the different sites in which thesensors are inserted. In another illustrative embodiment, the processorincludes an algorithm which provides a computational comparison betweena plurality of sensors including at least one sensor having elementscoated with an oxidoreductase such as glucose oxidase and at least onesensor not coated with glucose oxidase (e.g. which functions to identifybackground or interfering signals unrelated to blood glucose) in orderto, for example subtract signals unrelated to blood glucose and in thisway provide optimized sensor outputs. Certain embodiments of theinvention include apparatuses capable of multiplexing the signalsreceived from the plurality of sensors, e.g. by weaving multiple sensorsignals onto a single channel or communications line. In suchmultiplexing embodiments of the invention, segments of information fromeach signal can be interleaved and separated by time, frequency, orspace in order to obtain a comparative and comprehensive reading of allsensor outputs. Certain multiplexing embodiments of the inventioninclude a processor which uses an algorithm to provide computationalcomparisons between signals received from the plurality of sensors (e.g.to provide a mean, average or normalized value for the sensor signals).

Embodiments of the invention that include a plurality of sensors such asthose disclosed above can be used in methods designed to diminish orovercome problems associated with inserting a foreign body into thetissues of a patient (e.g. an immune response) and/or the variableanatomical features that can be encountered at a given insertion site(e.g. dermal layers that are of different thicknesses and/or depths indifferent individuals, the presence, absence or extent of adipose tissuedeposits, the presence of preexisting scar tissue and the like),phenomena which can effect the hydration and initialization of a sensor.In particular, patient physiologies which include for example highlyvariable and unpredictable localized immune responses at the insertionsite can cause problems with implantable sensors. For example, when aforeign object is implanted inside the body, one way the immune systemcan respond is to “wall off” the object in a fibrotic layer (e.g. sothat it is encapsulated in a fibrotic capsule), a phenomena which cancompromise the performance of an analyte sensor by inhibiting theability of an in vivo analyte (e.g. glucose) from contacting the sensor(due to the fibrotic layer acting as a physical barrier). In addition, apatient's anatomical characteristics which include for example highlyvariable and unpredictable tissue properties at different insertionsites can cause problems with implantable sensors which can include forexample different rates of analyte diffusion from the bloodstream to thesite an implanted sensor, a phenomena which can compromise theperformance of an analyte sensor by inhibiting the ability of a sensorto obtain relatively fast and/or accurate readings of blood glucoseconcentrations (e.g. a current or “real time” reading).

In typical method and device embodiments of the invention, the sensorsin such apparatuses are separated by a distance selected to be greatenough that the physiological response at a first insertion site for afirst sensor(s) does not influence the signal generated at a firstinsertion site for a first sensor(s). In addition, in certainembodiments of the invention, the sensors in such apparatuses areseparated by a distance great enough to allow the sensors to sense fromseparate tissue envelopes. For example, in one embodiment of theinvention, the constellation of elements in the apparatus is arranged sothat when the first and second piercing members are inserted into apatient, the first and second sensors are separated by at least 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, or 25 millimeters of tissue. In anotherembodiment of the invention, the constellation of elements in theapparatus is arranged so that when the first and second piercing membersare inserted into a patient, a first sensor is disposed within a layerof a first anatomical feature and/or characteristic such as aninterstitial space (i.e. the small, narrow spaces between tissues orparts of an organ epidermis) and a second sensor is disposed within alayer of a second anatomical feature and/or characteristic such in atissue or part of an organ. In one illustrative embodiment, a firstsensor is disposed within the epidermis and a second sensor is disposedwithin the dermis. In another embodiment of the invention, a firstsensor is disposed within adipose tissue and a second sensor is disposedwithin muscle tissue. In some embodiments of the invention, the piercingmembers are disposed on a base in a parallel configuration.Alternatively, the piercing members are disposed on a base in anon-parallel configuration (e.g. to facilitate a first sensor beingdisposed in a first anatomical feature and a second sensor is disposedwithin a second anatomical feature). Optionally, at least two sensors inan embodiment of the invention are disposed at about the same depthwithin the tissue in which they are implanted. Alternatively, at leasttwo sensors in an embodiment of the invention are disposed at differentdepths within the tissue in which they are implanted.

Embodiments of the invention that include a plurality of sensors such asthose noted above can overcome a variety of problems observed to occurwith single sensors by providing multiple physiological characteristicreadings at multiple insertion sites in a manner that compensates for orovercomes an occurrence of one or more of the above noted problems at asingle insertion site. For example, by using embodiments of theinvention constructed to include these elements, an immune response orproblematical anatomical feature (e.g. scar tissue) at a singleinsertion site will not compromise the function of the multiple sensorapparatus in view of the multiple/redundant sensor signals that areprovided by such embodiments of the invention. In addition, embodimentsof the invention that include a plurality of sensors can use themultiple sensor signals to characterize, compensate for and overcomeproblems associated with “drift” that can occur with a single sensor ata single insertion site (i.e. the phenomena where an output signal ofthe sensor changes over time independent of the measured property).

Embodiments of the invention that include a plurality of sensors can becombined with other sensor elements and/or configurations disclosedherein in order to further optimize sensor function and can comprise forexample electrodes distributed in a configuration that enhances theflexibility of the sensor structure and/or facilitates hydration of thesensor electrodes. Similarly, these embodiments of the invention can becombined with the apparatuses and methods that use voltage switchingand/or pulsing as part of the initialization and/or sensing process asdiscussed in detail below.

As noted above, certain embodiments of the invention can use voltageswitching or any other varying of voltage as part of the sensingprocess. As opposed to prior art methods, the voltage switching andstepping described herein is used to directly track glucose, instead offor hydration, initialization, or other purposes. Typical prior artmethods of continuous glucose monitoring use a fixed potential mode ofamperometry which returns a single current signal response. Therefore asingle measurement correlates to body glucose or other analytes. Unlikein prior art methods, which have fixed time period maximums, for example30 minutes over which varied voltages were used, in preferredembodiments, the present invention uses continuously changed voltageover the entire sensor period. In the prior art, 1 or 2 outputs wereused to track glucose changes, usually current output or voltage output.By varying voltage, many more parameters may be used, as discussedbelow.

In embodiments of the invention, varied voltage is used, for exampleapplying repeated cycles of step electrode potentials. The variedvoltage results in a continuous mode of glucose sensing providing muchmore information during chronological glucose monitoring. Using avarying voltage scheme such as a stepped voltage scheme has manyadvantages. For example, its inherent self-correlation is quite largecompared to a constant potential approach.

When step electrode potentials are used, for example each waveform cycleof signal relaxation response that is obtained contains a number ofcharacteristic electrode current readings (Isigs). These readings changeand relaxation times will directly correlate with glucoseconcentrations. Continuous repetition of such cycles results in a robustcontinuous glucose monitoring system. The characteristic signalresponses, by correlating to glucose, also correlate with each otherunder normal conditions throughout any glucose changes. Thus, thismethod provides higher system reliability as compared to a fixedpotential sensing mode, which only returns one characteristic electrodecurrent reading during sensing. Changes in system self-correlation basedon multiple electrode potentials can also be useful in identifying thepresence of substances that may interfere with glucose response andtracking such as interferents. In embodiments of the invention, multipleelectrode potentials are used, for example, stepped electrodepotentials.

In an embodiment of the present invention, a first electrode potentialis applied to an analyte sensor for a first predetermined period of timeand second electrode potential is applied to the analyte sensor for asecond predetermined period of time. The predetermined periods of timemay be the same or different. The application of the first and secondelectrode potentials are then reapplied, preferably for the samepredetermined periods of time as when originally applied. This cycle iscontinuously repeated throughout the sensing process, for a sensorduration time period. It is also possible to have more than twoelectrode potentials applied. For example, each cycle could have 3, 4, 5or even more voltages. Then, the cycle using all those voltages would becontinuously repeated over the sensor duration time period. The sensorduration time period preferably is the entire time that the sensor isimplanted in the body and being used for analyte sensing. If a hydrationand/or other run-in period such as for initialization is used, thesensor duration time period preferably starts after that run-in periodunless the same first and second electrode potentials are used duringthat period as well. If the same electrode potentials are used forrun-in and sensing, the sensor duration time period could include therun-in time, but it is usually better to start the sensor duration timeperiod after any run-in that could affect sensor values. In furtherembodiments, the voltage switching of the invention could last for adifferent period of time, for example, for the time it takes to get oneor a predetermined number of analyte readings. In certain embodiments,the sensor time period lasts throughout the entire sensing period of asensor, until the sensor is disconnected from its electronics,physically or by the electronics being otherwise turned off such that nocurrent is being applied to the sensor. The sensor duration time periodmay be greater than 5 minutes, greater than 10 minutes, greater than 15minutes, greater than 30 minutes, greater than an hour, greater than 3hours, or even more time, such as a day.

As discussed herein, methods such as voltage switching may be used toinitialize the sensor prior to the time at which sensing data will beused to determine analyte readings. As such, there may be aninitialization period prior to the sensor duration time period. Inaddition or alternatively, also as discussed herein, there may be ahydration period prior to the sensor duration time period.

As discussed herein, the sensor may be directly connected to sensorelectronics, which may be part of or separately connected (wirelessly orvia or other direct connection) to a monitoring device that monitors thesignals received from the sensor. Depending on the construction of thesensor device and/or monitor (whether separate or together with thesensor device), one or both of the sensor electronics and monitor maymake calculations based on the signals sensed at the sensor to convertthe signals to actual analyte measurements and to determine variouscharacteristics of the data received. As discussed herein, a number ofdifferent characteristics may be used to help get a more accuratepicture of the actual level of analyte in the patient. Thesecharacteristics can include current values at different time intervals,such as during relaxation of the curve, change in currents, change intotal charge, and/or calculated relaxation parameters.

FIGS. 4A and 4B demonstrate some of the information that can be used toevaluate analyte concentration using a stepped potential method. In FIG.4A, a graph is shown with the Y-axis representing potential andelectrode current reading (Isig) and the X-axis representing time. Inthis embodiment, there are two stepped potentials 401 and 402. Thepotentials are repeated. The Isig readings are shown by the dotted line403. As can be seen in FIG. 4A, the Isig readings change during time,giving a varying reading. This is unlike what occurs in a singlepotential system, which would have only one, flat Isig reading. FIG. 4Bhas been labeled to display some example portions of the Isig curvesthat can be correlated to analyte levels. For example, characteristicIsig readings 405 can be used. Shown in FIG. 4B are the high and lowreadings (a′, c′, d′ and f′). Also shown are the valleys of the curves(b′ and e′). Another type of reading is the change in Isig during avoltage application or between voltage applications. These are shown inFIG. 4B by 406 (a, b, c, d, and e). The kinetics of the relaxationcurves can be used (407) and the characteristic area under therelaxation curves (408, also known as the total charge transferred) canbe used.

To measure the kinetics of the relaxation curves, a simple electrodeequation can be used. FIGS. 5A and 5B show graphs illustratingproperties for linear diffusion in a simple electrode system. FIG. 5Aillustrates the concentration profiles for linear diffusion in asolution where no electric current has been applied and there is nostirring. As can be seen, the concentration profile at the distance fromthe surface changes from a logarithmic type profile to a linear profileas time increases. In FIG. 5B, the voltage applied was 0.535V. Thenormalized current (X-axis) approaches zero as time (Y-axis) increases.The decrease of current (Isig) can be modeled, for example, by thefollowing Cottrel Equation:

${i(t)} = \frac{{nFAD}^{1\text{/}2}C_{0}}{\pi^{1\text{/}2}t^{1\text{/}2}}$where i=current, n=# of electrons, F=Faraday constant, A=surface area,D=diffusion constant, C₀, and t=time. Other equations that model theIsig profile can also be used to estimate Isig at a particular time. Forexample, to include boundary layer effects, the following equation maybe used:

$\begin{matrix}{{i(t)} = {\frac{{nFADC}_{R}}{d}\left\lbrack {1 + {2{\sum\limits_{n = 1}^{\infty}\;{\mathbb{e}}^{{- \frac{n^{2}\pi^{2}D}{d^{2}}}t}}}} \right\rbrack}} & {{equation}\mspace{14mu} 1}\end{matrix}$where i=current, n=# of electrons, F=Faraday constant, A=surface area,D=diffusion constant, C_(r)=concentration, d=boundary layer distance,and t=time.

To model the sensor and the solution, a 2 component model may be used.For example, a combination of the Cottrell equation and boundary layerconditions can be used. The Cotrell equation captures aspects of thesensor that act as a pure electrode system with no stirring. Theboundary layer component treats the layers of the sensors as part of theboundary with an effective diffusion constant. An example equationcombining these models is:

$\begin{matrix}{{i(t)} = {{\frac{{nFADC}_{R}}{d}\left\lbrack {1 + {2{\sum\limits_{n = 1}^{\infty}\;{\mathbb{e}}^{{- \frac{n^{2}\pi^{2}D}{d^{2}}}t}}}} \right\rbrack} + \frac{{nFAD}^{1\text{/}2}C_{0}}{\pi^{1\text{/}2}t^{1\text{/2}}}}} & {{equation}\mspace{14mu} 2}\end{matrix}$

FIGS. 6A-C are three graphs showing a waveform fitted using the aboveequation, equation 2. FIG. 6A shows the measured current from a firstvoltage −535V and a second voltage 535V. The second graph, FIG. 6B showsthe measured current 501 and the calculated current 502 for the firstvoltage. As can be seen, the calculated current correlates very wellwith the current as actually measured. FIG. 6C shows the measuredcurrent 501 and calculated current 502 for the second voltage.

Another equation that can be used to model the sensor values has fourmain components (P1-P4) and is as follows: (parameters are as above,with α and β each being a weighting factor to indicate how much theweighted equation is being taken into account. If each is weightedequally, α and β each equal 1, but they may be adjusted as desired):

$\begin{matrix}{{i(t)} = {{a\begin{matrix}~ \\\frac{{nFADC}_{R}}{d} \\{P\; 1}\end{matrix}{\mathbb{e}}\begin{matrix}~ \\\ ^{{- \frac{n^{2}\pi^{2}D}{d^{2}}}t} \\{P\; 2}\end{matrix}} + \begin{matrix}~ \\\frac{{nFAD}^{1\text{/}2}C_{0}}{\pi^{1\text{/}2}t^{1\text{/}2}} \\{P\; 3}\end{matrix} + {\beta\begin{matrix}~ \\\frac{{nFADC}_{R}}{d} \\{P\; 4}\end{matrix}}}} & {{equation}\mspace{14mu} 3}\end{matrix}$The parameters P1-P4 can be used in combination or independently totrack sensor glucose as well as sensor performance. As with the otherequations disclosed herein or that may otherwise be used for trackingsensor glucose and/or sensor performance, the monitor, sensorelectronics, or other device that receives signals and/or data from thesensor would calculate the parameters based on the sensor signals.Aspect of the equations noted above are well known in the art anddiscussed, for example, in Bard, A. J.; Faulkner, L. R. “ElectrochemicalMethods. Fundamentals and Applications” 2nd Ed. Wiley, New York. 2001and F. G. Cottrell Zeitschrift Fur Physikalische Chemie, 42:385, 1902.

Calculations based on the above equation, equation 3, include a numberof ratios to determine sensor performance. For example, the ratio P1/P2gives a constant times C_(R)d, indicating that varying CR(concentration) may give d (the boundary layer distance). The size of dwill indicate if the relaxation calculated truly describes an area thatis “within” the sensor itself or is actually outside the sensor area.For example, a d value of less than about 9 microns may indicate thesensor and greater than about 9 microns may indicate in the solution.The ratio of P1/P3, assuming that diffusion constants and concentrationsare proportional, gives information about the diffusion of analyte(e.g., glucose) through the system versus the distance the analytediffuses. Thus, the ratio of P1/P3 gives information that may be usedfor sensor diagnostics. The ratio of P1/P4 is the ratio of α/β and canbe used to check boundary layer stability, for example during dynamiccheck vs. steady state (hinting as to whether or not diffusioncharacteristics look different from steady state characteristics). Inembodiments, P1/P4 trends towards about 1 for good production ofsensors.

Properly functioning or “good” and improperly functioning or “bad”sensors have characteristically different behaviors such that whenpulsed voltage schemes are used that the sensor chemistry can bedistinguished. Good sensors are those that are working properly and donot have any problems that would significantly affect their readings.Problems that could create a bad sensor, which would not measureanalytes accurately or precisely, could include damage, improperchemistry deposition, poor connections between electrodes and contactpads, and any other problems that affect sensor readings. In addition,sensors can go bad over time. For example, there may be a change in theenvironment of the sensor from a number of biological factors such asimmunological responses. These types of changes could cause the sensorsensitivity to degrade, creating a bad sensor. By using the parametersin the above equations, it is possible to determine whether suchdegradation is occurring. Replacement of the bad sensor with a goodsensor or recalculation based on the degradation could be then done tocorrect the readings. There could be a prompt to the user to replace thesensor on the monitor, sensor housing, or other device communicatingwith the sensor.

The voltage switching/varying is preferably continuous after the sensorhas been initialized until the sensing of that particular sensor hasbeen completed. This could be, for example, 3 days for a 3-day sensor.If the sensor monitor and/or other sensor electronics are disconnectedfrom the sensor for showers, exercise such as swimming, or otherreasons, the voltage switching may be discontinued and started againupon reconnection after any necessary initialization.

Although the figures show the use of two voltages, it is also possibleto use more voltages, such as 3, 4, 5 or even more. These voltages maybe stepped voltages. For example, the steps may be structured such thateach successive voltage in a cycle of voltages is higher or lower thanthe previous voltage. The steps of voltage would then begin again at thefirst voltage, with the cycle continuing during sensing. The voltagesmay be equal in distance from each other. For example, voltage 1 couldbe 600 mv and voltage 2 could be 535 mv. If there was a third voltagethat is equal in distance, it would be 470 mv. A fourth voltage would be405 mv, and so on. Alternatively, the voltages could be varied indistance from each other. An example of this would be: voltage 1=600 mv;voltage 2=535 mv; voltage 3=500 mv. It is also possible that the 3^(rd)voltage could be higher in this scheme as in the following example:voltage 1=600 mv; voltage 2=535 mv; voltage 3=550 mv.

In further embodiments, a negative voltage is applied as one or more ofthe voltages. One example is where voltage 1 is 535 mv and voltage 2 is−535 mv. Another example of varied voltages is as follows: voltage 1=535mv; voltage 2=177 mv. In one illustrative embodiment, the sensor isswitched between a first potential such as −535, 0, 177, 280, 535, 635or 1.070 millivolts and a second potential such as −535, 0, 177, 280,535, 635 or 1.070 millivolts. Other voltages may be used as desired.

The voltages are preferably applied for predetermined periods of time.The application of a voltage can be, for example, 1, 3, 5, 7, 10, 15,30, 45, 60, 90 or 120 seconds. Different time periods may be used aswell. The time each voltage is applied can be the same or different asthe other voltage(s). In embodiments, each cycle has each voltageapplied for the same predetermined period of time as in the previousvoltage, but in more complicated methods, the voltages could be appliedfor different times in different cycles or in groups of differentcycles.

In further embodiments of the invention, a pulsed (e.g. produced ortransmitted or modulated in short bursts or pulses) voltage can be usedinstead of cycles of voltages for longer periods of time. Such pulsingfor example can be used to reduce/compensate for background currentreadings. Pulsing allows sensors to detect lower concentrations ofglucose more efficiently, that there is a linear response to glucoseswitching, and that pulsing can be used to both decrease the backgroundcurrent and reduce the effect of interferents. Sensor systems caninclude a processor including software algorithms that control factorssuch as voltage output and/or working potential and/or pulsing and orswitching and/or the time periods of such factors. Sensor systems canalso include various hardware features designed to facilitate voltagepulsing, for example discharge circuit elements. For example, highfrequency switching can require a discharge circuit element so thatlayers discharge held charge (wherein the sensor layers analogous to acapacitor). One illustrative embodiment is sensor having two specificpotential dedicated electrodes (e.g. at 280 mv and 535 mv) and isdesigned to obtain readings of both electrodes as sensor switchesbetween them. In this context, it is known in art to take sensor readingat a wide range of potentials (see, e.g. U.S. Pat. Nos. 5,320,725,6,251,260, 7,081,195 and Patent Application No. 2005/0161346).

In further embodiments of the invention, a sensor may function byapplying a first voltage for a first time, by optionally waiting apredetermined period of time (i.e., not applying a voltage), and thencycling between the application of the first voltage and the waiting ofa predetermined period of time for a number of iterations or a specifictimeframe. A pair of voltages may be applied to create an anodic cycleand then a cathodic cycle. The first voltage may have a positive valueor a negative value. The second voltage may have a positive value ornegative value. Under certain operating conditions, a voltage magnitudeof the first voltage for one of the iterations may have a differentmagnitude from a voltage magnitude of the first voltage for a second ordifferent iteration. In an embodiment of the invention, a voltagewaveform, such as a ramp waveform, a stepped waveform, a sinusoidwaveform, and a squarewave waveform, may be applied as the firstvoltage. Any of the above mentioned waveforms may also be applied as thesecond voltage. Under certain operating conditions, the voltage waveformapplied as the first voltage in a first iteration may differ from thevoltage waveform applied as the first voltage in the second iteration.The same may hold true for the application of the second voltage. Undercertain operating conditions, a voltage waveform may be applied as thefirst voltage to the sensor and a voltage pulse may be applied as thesecond voltage to the sensor.

A plurality of short duration voltage pulses may be applied for a firsttimeframe to initiate the anodic cycle in the sensor and a plurality ofshort duration voltage pulses may be applied for a second timeframe toinitiate the cathodic cycle in the sensor. The magnitude of the firstplurality of short duration pulses may be different from the magnitudeof the second plurality of short duration pulses. In an embodiment ofthe invention, the magnitude of some of the pulses in the firstplurality of short duration pulses may have different values from themagnitude of other pulses in the first plurality of short durationpulses. The shorter duration voltage pulses may be utilized to apply thefirst voltage, the second voltage, or both. In an embodiment of thepresent invention, the magnitude of the shorter duration voltage pulsefor the first voltage is −1.07 volts and the magnitude of the shorterduration voltage pulse for the second voltage is approximately half ofthe high magnitude, e.g., −0.535 volts. Alternatively, the magnitude ofthe shorter duration pulse for the first voltage may be 0.535 volts andthe magnitude of the shorter duration pulse for the second voltage is1.07 volts.

In embodiments of the invention utilizing short duration pulses, thevoltage may be applied not continuously for the entire first timeperiod. Instead, in the first time period, the voltage applicationdevice may transmit a number of short duration pulses during the firsttime period. In other words, a number of mini-width or short durationvoltage pulses may be applied to the electrodes of the sensors over thefirst time period. Each mini-width or short duration pulse may a widthof a number of milliseconds. Illustratively, this pulse width may be 30milliseconds, 50 milliseconds, 70 milliseconds or 200 milliseconds.These values are meant to be illustrative and not limiting.

In another embodiment of the invention, each short duration pulse mayhave the same time duration within the first time period. For example,each short duration voltage pulse may have a time width of 50milliseconds and each pulse delay between the pulses may be 950milliseconds. In this example, if two minutes is the measured time forthe first timeframe, then 120 short duration voltage pulses may beapplied to the sensor. In an embodiment of the invention, each of theshort duration voltage pulses may have different time durations. In anembodiment of the invention, each of the short duration voltage pulsesmay have the same amplitude values. In an embodiment of the invention,each of the short duration voltage pulses may have different amplitudevalues. By utilizing short duration voltage pulses rather than acontinuous application of voltage to the sensors, the same anodic andcathodic cycling may occur and the sensor (e.g., electrodes) issubjected to less total energy or charge over time. The use of shortduration voltage pulses utilizes less power as compared to theapplication of continuous voltage to the electrodes because there isless energy applied to the sensors (and thus the electrodes).

Embodiments of the invention can use voltage switching not only in thedetection of interfering species and/or specific analyte concentrationsbut also to facilitate the hydration and/or initialization of varioussensor embodiments of the invention. In particular, the time forinitialization (“run-in”) differs for different sensors and can takehours. Embodiments of the invention include a sensor initializationscheme involving high frequency initialization (switching of voltagepotentials). In one illustrative embodiment, a triple initializationprofile is used where the voltage of the sensor is switched between afirst potential such as 0, 280, 535, 635 or 1.070 millivolts and asecond potential such as 0, 280, 535, 635 or 1.070 millivolts over aperiod of 5, 10, 20, 30 or 45 seconds or 1, 5, 10 or 15 minutes. Certainvoltage switching embodiments of the invention further use voltagepulsing in the detection of analyte signals. The number of pulses usedin such embodiments of the invention is typically at least 2 and can be3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more. Pulses can be for apredetermined period of time, for example 1, 3, 5, 7, 10, 15, 30, 45,60, 90 or 120 seconds. One illustrative example of this comprises 6pulses, each a few seconds long. By using such embodiments of theinvention, the sensor run-in is greatly accelerated, a factor whichoptimizes a user's introduction and activation of the sensor. Certain ofthese methods can be adapted for use with similar methods known in theart (see, e.g. U.S. Pat. Nos. 5,320,725; 6,251,260 and U.S. PatentApplication No. 2005/0161346, the content of which are incorporated byreference). Further discussion of voltage switching for initializationand hydration can be found in U.S. patent application Ser. Nos.12/184,046 (filed Jul. 31, 2008), 11/322,977 (filed Dec. 30, 2005), and11/323,242 (filed Dec. 30, 2005), which are herein incorporated byreference.

Some embodiments of the invention include a fuse element that can betriggered after a predetermined period of time or event so as tointerrupt a flow of electrical current within the apparatus (i.e. so asto disable the sensor). For example, one embodiment of the inventionincludes a sensor operatively coupled to a sensor input capable ofreceiving a signal from the sensor that is based on a sensedphysiological characteristic value in the mammal; and a processorcoupled to the sensor input, wherein the processor is capable oftriggering a fuse element to disable the sensor after a predeterminedperiod which is based upon the in vivo lifetime of the sensor. Furtherdisclosure of sensors that use a fuse element can be found in U.S.patent application Ser. No. 12/184,046 (filed Jul. 31, 2008), which isherein incorporated by reference.

In some embodiments of the invention, a processor is capable ofcomparing a first signal received from a working electrode in responseto a first working potential with a second signal received from aworking electrode in response to a second working potential, wherein thecomparison of the first and second signals at the first and secondworking potentials can be used to identify a signal generated by aninterfering compound. In one such embodiment of the invention, oneworking electrode is coated with glucose oxidase and another is not, andthe interfering compound is acetaminophen, ascorbic acid, bilirubin,cholesterol, creatinine, dopamine, ephedrine, ibuprofen, L-dopa,methyldopa, salicylate, tetracycline, tolazamide, tolbutamide,triglycerides or uric acid. Optionally, a pulsed and/or varied (e.g.switched) voltage is used to obtain a signal from a working electrode.Typically, at least one voltage is 280, 535 or 635 millivolts. Relatedembodiments of the invention include methods for identifying and/orcharacterizing one or more signals generated by an interfering compoundin various sensor embodiments of the invention (e.g. by comparing thesignal from an electrode coated with an analyte sensing compound with acomparative electrode not coated with an analyte sensing compound).These methods are further discussed in U.S. application Ser. Nos.12/184,046 (filed Jul. 31, 2008), which is herein incorporated byreference.

In a related embodiment of the invention, a processor compares a firstsignal received from a working electrode coated with glucose oxidase inresponse to a first working potential with a second signal received froma working electrode coated with glucose oxidase in response to a secondworking potential, wherein the comparison of the first and secondsignals at the first and second working potentials is used tocharacterize a blood glucose concentration within at least one discreetconcentration range. In certain embodiments of the invention at leasttwo working potentials of approximately 280, 535 or 635 millivolts isused. In some embodiments of the invention, the comparison of the firstand second signals at the first and second working potentials can beused to characterize a blood glucose concentration within aconcentration range below 50 or 70 mg/dL (i.e. values typicallyassociated with hypoglycemia) or above 125, or 150 mg/dL (i.e. valuestypically associated with hyperglycemia). In certain embodiments of theinvention a 280 mv potential is used because it can detect lowerconcentrations of glucose more efficiently. Related embodiments of theinvention include methods for identifying and/or characterizing aspecific analyte concentration or range of analyte concentrations usingthe various sensor embodiments of the invention (e.g. by comparing theanalyte signal from one or more electrodes at different workingpotentials, wherein the different working potentials are selected fortheir ability to characterize a specific analyte concentration and/orrange of analyte concentrations).

In another illustrative embodiment of the invention, the processor iscapable of characterizing a plurality of signals received from thesensor by for example comparing a first signal received from a workingelectrode coated with glucose oxidase with a second signal received froma working electrode not coated with glucose oxidase so as to obtaininformation on a background signal that is not based on a sensedphysiological characteristic value in the mammal. In anotherillustrative embodiment of the invention, the processor is capable ofcharacterizing a plurality of signals received from the sensor bycomparing a first signal received from a working electrode coated withglucose oxidase with a second signal received from a working electrodenot coated with glucose oxidase so as to obtain information on a signalgenerated by an interfering compound. In another embodiment of theinvention, two working electrodes are coated with glucose oxidase andthe processor is capable of obtaining information on glucoseconcentrations in the mammal by comparing the signals received from thetwo working electrodes coated with glucose oxidase.

Certain sensor embodiments switch between a high potential to a lowpotential (e.g. with a frequency of less than 3, 2 or 1 seconds). Insuch embodiments, a sensor may not discharge, with for example sensorelements acting as a sort of capacitor. In this context, someembodiments of the invention can include a circuit discharge elementthat facilitates sensor circuit discharge (e.g. if discharge is notsufficient to reach a specific potential such as 535 millivolts). Avariety of such circuit discharge elements known in the art can beadapted for use with sensor embodiments of the invention (see, e.g. U.S.Pat. Nos. 4,114,627; 4,373,531; 4,858,610; 4,991,583; and 5,170,806,5,486,201, 6,661,275 and U.S. Patent Application No. 20060195148).Optionally for example, a sensor charge can be removed by connecting itthrough a discharging switch element, and optionally a dischargingresistor element.

Certain embodiments of the invention include a processor that detectswhether a sensor is sufficiently hydrated for analyte detectioncomprising a computer usable media including at least one computerprogram embedded therein that is capable of calculating an impedancevalue; and comparing the impedance value against a threshold todetermine if the sensor is sufficiently hydrated for analyte detection.A related embodiment of the invention is a method of detecting whether asensor is sufficiently hydrated for analyte detection, comprisingcalculating an open circuit potential value between at least twoelectrodes of the sensor; and comparing the open circuit potential valueagainst a threshold to determine if the sensor sufficiently hydrated foranalyte detection. Typically, the open circuit potential value is theimpedance value (and optionally this value is an approximation of a sumof polarization resistance and solution resistance). Optionally, theopen circuit potential value is compared against an another threshold todetermine if the sensor sufficiently hydrated for analyte detection.This can solve problems that occur when a user attempts to initialize asensor that is not fully hydrated (e.g. compromising the accuracy and/orlifetime of the sensor).

Certain embodiments of the invention include materials that facilitatethe use of glucose oxidase, by for example, incorporating materials thatfunction to optimize the stoichiometry of a reaction of interest (e.g.to overcome the oxygen deficit problem). Optionally for example, theanalyte sensing layer comprises an oxidoreductase that generateshydrogen peroxide upon exposure to a ligand for the oxidoreductase,wherein the amount of hydrogen peroxide generated by the polypeptide isproportional to the amount of ligand exposed to the polypeptide.Typically, the oxidoreductase polypeptide comprises an enzyme selectedfrom the group consisting of glucose oxidase, glucose dehydrogenase,lactate oxidase, hexokinase and lactose dehydrogenase.

As noted above, it has been discovered that certain crosslinkingreagents can be used for example to produce crosslinked polypeptidelayers having a constellation of structural and chemical properties thatmake them surprisingly useful in certain contexts (e.g. when used tocrosslink carrier proteins such albumin and enzymes such as glucoseoxidase within a layer of a sensor apparatus having a plurality ofoverlapping functional layers). As is known in the art, crosslinking isthe process of chemically joining two or more molecules by a covalentbond. Crosslinking compounds typically comprise a linker “arm” thatfunctions as a tether between crosslinked compounds as well as at leasttwo chemical moieties (typically on distal ends of the arm of thecompound) that react specific functional groups on proteins or othermolecules (see, e.g. primary amines, sulfhydryls and the like).Illustrative crosslinking compounds are shown, for example in U.S.patent application Ser. No. 12/184,046, which is herein incorporated byreference.

As noted above, embodiments of the invention include methods for makingthe sensor embodiments disclosed herein. Certain methods for making thesensor embodiments disclosed herein include the step of preciselycontrolling the concentration of a constituent so as to effect itsmorphology, function or the like. For example in sensors that use GOx, aconcentration range of about 20-40 KU (and 5% Human Serum Albumin) canbe used to optimize GOx layer morphology. Methods for making the sensorembodiments disclosed herein include the step of applying anoxidoreductase (e.g. a GOx composition) onto the surface of an electrodevia brushing methods that facilitate its disposal in proximity toreactive surface. In this context, brushing (e.g. with the equivalent ofa tiny paintbrush) GOx onto electrode surface and/or writing GOx ontoelectrode surface using a pen-type device can be employed rather thandepositing a droplet of the solution, a procedure which (e.g. due tosurface tension of droplet) can produce uneven deposition. Moreover,such brushing steps can push a composition solution deep into theconvoluted reactive surface of a Pt black of electrode. In addition,brushing is easier than processes such as spin coating because it allowsfor a more precise localized deposition of a composition. In thiscontext, brushing allows for example, the easy coating of small reactivesurfaces that are not amenable to coating by other means (e.g. pipettingand/or spin coating processes). Certain embodiments for making theinvention can be performed under a vacuum to, for example, pull out airand facilitate application of a layer to a substrate.

As noted above, the sensor embodiments disclosed herein can be used tosense analytes of interest in one or more physiological environments. Incertain embodiments for example, the sensor can be in direct contactwith interstitial fluids as typically occurs with subcutaneous sensors.The sensors of the present invention may also be part of a skin surfacesystem where interstitial glucose is extracted through the skin andbrought into contact with the sensor (see, e.g. U.S. Pat. Nos. 6,155,992and 6,706,159 which are incorporated herein by reference). In otherembodiments, the sensor can be in contact with blood as typically occursfor example with intravenous sensors. The sensor embodiments of theinvention further include those adapted for use in a variety ofcontexts. In certain embodiments for example, the sensor can be designedfor use in mobile contexts, such as those employed by ambulatory users(e.g. a diabetic performing daily activities). Alternatively, the sensorcan be designed for use in stationary contexts such as those adapted foruse in clinical settings. Such sensor embodiments include, for example,those used to monitor one or more analytes present in one or morephysiological environments in a hospitalized patient (e.g. a patientconfined to a hospital bed in situations such as those described in WO2008042625).

Sensors of the invention can also be incorporated in to a wide varietyof medical systems known in the art. Sensors of the invention can beused, for example, in a closed loop infusion systems designed to controlthe rate that medication is infused into the body of a user. Such aclosed loop infusion system can include a sensor and an associated meterwhich generates an input to a controller which in turn operates adelivery system (e.g. one that calculates a dose to be delivered by amedication infusion pump). In such contexts, the meter associated withthe sensor may also transmit commands to, and be used to remotelycontrol, the delivery system. Typically, the sensor is a subcutaneoussensor in contact with interstitial fluid to monitor the glucoseconcentration in the body of the user, and the liquid infused by thedelivery system into the body of the user includes insulin. Illustrativesystems are disclosed for example in U.S. Pat. Nos. 6,558,351 and6,551,276; PCT Application Nos. US99/21703 and US99/22993; as well as WO2004/008956 and WO 2004/009161, all of which are incorporated herein byreference.

Certain embodiments of the invention measure peroxide and have theadvantageous characteristic of being suited for implantation in avariety of sites in the mammal including regions of subcutaneousimplantation and intravenous implantation as well as implantation into avariety of non-vascular regions. A peroxide sensor design that allowsimplantation into non-vascular regions has advantages over certainsensor apparatus designs that measure oxygen due to the problems withoxygen noise that can occur in oxygen sensors implanted intonon-vascular regions. For example, in such implanted oxygen sensorapparatus designs, oxygen noise at the reference sensor can compromisethe signal to noise ratio which consequently perturbs their ability toobtain stable glucose readings in this environment. The sensors of theinvention therefore overcome the difficulties observed with such oxygensensors in non-vascular regions.

Certain sensor embodiments of the invention further include advantageouslong term or “permanent” sensors which are suitable for implantation ina mammal for a time period of greater than 30 days. In particular, as isknown in the art (see, e.g. ISO 10993, Biological Evaluation of MedicalDevices) medical devices such as the sensors described herein can becategorized into three groups based on implant duration: (1) “Limited”(<24 hours), (2) “Prolonged” (24 hours-30 days), and (3) “Permanent”(>30 days). In some embodiments of the invention, the design of theperoxide sensor of the invention allows for a “Permanent” implantationaccording to this categorization, i.e. >30 days. In related embodimentsof the invention, the highly stable design of the peroxide sensor of theinvention allows for an implanted sensor to continue to function in thisregard for 2, 3, 4, 5, 6 or 12 or more months.

Typically, the analyte sensor apparatus includes an analyte sensinglayer disposed on a conductive layer of the sensor, typically covering aportion or all of the working electrode. This analyte sensing layerdetectably alters the electrical current at the working electrode in theconductive layer in the presence of an analyte to be sensed. Asdisclosed herein, this analyte sensing layer typically includes anenzyme or antibody molecule or the like that reacts with the analyte ofinterest in a manner that changes the concentrations of a molecule thatcan modulate the current at the working electrode (see e.g. oxygenand/or hydrogen peroxide as shown in the reaction scheme of FIG. 1).Illustrative analyte sensing layers comprise an enzyme such as glucoseoxidase (e.g. for use in glucose sensors) or lactate oxidase (e.g. foruse in lactate sensors). In some embodiments of the invention, theanalyte sensing layer is disposed upon a porous metallic and/or ceramicand/or polymeric matrix with this combination of elements functioning asan electrode in the sensor. In certain embodiments of the invention thatrequire a robust design (e.g. long-term sensors), a ceramic base is usedas a dielectric (rather than a polyimide) due to its relatively strongermaterial properties.

Typically, the analyte-sensing layer further comprises a carrier proteinin a substantially fixed ratio with the analyte sensing compound (e.g.the enzyme) and the analyte sensing compound and the carrier protein aredistributed in a substantially uniform manner throughout the analytesensing layer. Typically the analyte sensing layer is very thin, forexample, less than 1, 0.5, 0.25 or 0.1 microns in thickness. While notbeing bound by a specific scientific theory, it is believed that sensorshaving such thin analyte sensing layers have surprisingly enhancedcharacteristics as compared to the thicker layers that are typicallygenerated by electrodeposition because electrodeposition produces 3-5micron thick enzyme layers in which only a fraction of the reactiveenzyme within the coating layer is able to access the analyte to besensed. Such thicker glucose oxidase pellets that are produced byelectrodeposition protocols are further observed to have a poormechanical stability (e.g. a tendency to crack) and further take alonger time to prepare for actual use, typically taking weeks of testingbefore it is ready for implantation. As these problems are not observedwith the thin layered enzyme coatings described herein, these thincoatings are typical embodiments of the invention.

Optionally, the analyte sensing layer has a protein layer disposedthereon and which is typically between this analyte sensing layer andthe analyte modulating layer. A protein within the protein layer is analbumin selected from the group consisting of bovine serum albumin andhuman serum albumin. Typically this protein is crosslinked. Withoutbeing bound by a specific scientific theory, it is believed that thisseparate protein layer enhances sensor function and provides surprisingfunctional benefits by acting as a sort of capacitor that diminishessensor noise (e.g. spurious background signals). For example, in thesensors of the invention, some amount of moisture may form under theanalyte modulating membrane layer of the sensor, the layer whichregulates the amount of analyte that can contact the enzyme of theanalyte sensing layer. This moisture may create a compressible layerthat shifts within the sensor as a patient using the sensor moves. Suchshifting of layers within the sensor may alter the way that an analytesuch as glucose moves through the analyte sensing layers in a mannerthat is independent of actual physiological analyte concentrations,thereby generating noise. In this context, the protein layer may act asa capacitor by protecting an enzyme such as GOx from contacting themoisture layer. This protein layer may confer a number of additionaladvantages such as promoting the adhesion between the analyte sensinglayer and the analyte modulating membrane layer. Alternatively, thepresence of this layer may result in a greater diffusion path formolecules such as hydrogen peroxide, thereby localizing it to theelectrode sensing element and contributing to an enhanced sensorsensitivity.

Typically, the analyte sensing layer and/or the protein layer disposedon the analyte sensing layer has an adhesion promoting layer disposedthereon. Such adhesion promoting layers promote the adhesion between theanalyte sensing layer and a proximal layer, typically an analytemodulating layer. This adhesion promoting layer typically comprises asilane compound such as γ-aminopropyltrimethoxysilane which is selectedfor its ability to promote optimized adhesion between the various sensorlayers and functions to stabilize the sensor. Interestingly, sensorshaving such a silane containing adhesion promoting layers exhibitunexpected properties including an enhanced overall stability. Inaddition, silane containing adhesion promoting layers provide a numberof advantageous characteristics in addition to an ability to enhancingsensor stability, and can, for example, play a beneficial role ininterference rejection as well as in controlling the mass transfer ofone or more desired analytes.

In certain embodiments of the invention, the adhesion promoting layerfurther comprises one or more compounds that can also be present in anadjacent layer such as the polydimethyl siloxane (PDMS) compounds thatserves to limit the diffusion of analytes such as glucose through theanalyte modulating layer. The addition of PDMS to the AP layer forexample can be advantageous in contexts where it diminishes thepossibility of holes or gaps occurring in the AP layer as the sensor ismanufactured.

Typically the adhesion promoting layer has an analyte modulating layerdisposed thereon which functions to modulate the diffusion of analytestherethrough. In one embodiment, the analyte modulating layer includescompositions (e.g. polymers and the like) which serve to enhance thediffusion of analytes (e.g. oxygen) through the sensor layers andconsequently function to enrich analyte concentrations in the analytesensing layer. Alternatively, the analyte modulating layer includescompositions which serve to limit the diffusion of analytes (e.g.glucose) through the sensor layers and consequently function to limitanalyte concentrations in the analyte sensing layer. An illustrativeexample of this is a hydrophilic glucose limiting membrane (i.e.functions to limit the diffusion of glucose therethrough) comprising apolymer such as polydimethyl siloxane or the like. In certainembodiments of the invention, the analyte modulating layer comprises ahydrophilic comb-copolymer having a central chain and a plurality ofside chains coupled to the central chain, wherein at least one sidechain comprises a silicone moiety.

Typically the analyte modulating layer further comprises one or morecover layers which are typically electrically insulating protectivelayers disposed on at least a portion of the sensor apparatus (e.g.covering the analyte modulating layer). Acceptable polymer coatings foruse as the insulating protective cover layer can include, but are notlimited to, non-toxic biocompatible polymers such as silicone compounds,polyimides, biocompatible solder masks, epoxy acrylate copolymers, orthe like. An illustrative cover layer comprises spun on silicone.Typically the cover layer further includes an aperture that exposes atleast a portion of a sensor layer (e.g. analyte modulating layer) to asolution comprising the analyte to be sensed.

The analyte sensors described herein can be polarized cathodically todetect, for example, changes in current at the working cathode thatresult from the changes in oxygen concentration proximal to the workingcathode that occur as glucose interacts with glucose oxidase as shown inFIG. 1. Alternatively, the analyte sensors described herein can bepolarized anodically to detect for example, changes in current at theworking anode that result from the changes in hydrogen peroxideconcentration proximal to the working anode that occur as glucoseinteracts with glucose oxidase as shown in FIG. 1. In typicalembodiments of the invention, the current at the working electrode(s) iscompared to the current at a reference electrode(s) (a control), withthe differences between these measurements providing a value that canthen be correlated to the concentration of the analyte being measured.Analyte sensor designs that obtain a current value by obtaining ameasurement from a comparison of the currents at these dual electrodesare commonly termed, for example, dual oxygen sensors.

In some embodiments of the invention, the analyte sensor apparatus isdesigned to function via anodic polarization such that the alteration incurrent is detected at the anodic working electrode in the conductivelayer of the analyte sensor apparatus. Structural design features thatcan be associated with anodic polarization include designing anappropriate sensor configuration comprising a working electrode which isan anode, a counter electrode which is a cathode and a referenceelectrode, and then selectively disposing the appropriate analytesensing layer on the appropriate portion of the surface of the anodewithin this design configuration. Optionally this anodic polarizationstructural design includes anodes, cathodes and/or working electrodeshaving different sized surface areas. For example, this structuraldesign includes features where the working electrode (anode) and/or thecoated surface of the working electrode is larger or smaller than thecounter electrode (cathode) and/or the coated surface of the counterelectrode (e.g. a sensor designed to have a 1× area for a referenceelectrode, a 2.6× area for a working electrode and a 3.6× area for acounter electrode). In this context, the alteration in current that canbe detected at the anodic working electrode is then correlated with theconcentration of the analyte. In certain illustrative examples of thisembodiment of the invention, the working electrode is measuring andutilizing hydrogen peroxide in the oxidation reaction (see e.g. FIG. 1),hydrogen peroxide that is produced by an enzyme such as glucose oxidaseor lactate oxidase upon reaction with glucose or lactate respectively.Such embodiments of the invention relating to electrochemical glucoseand/or lactate sensors having such hydrogen peroxide recyclingcapabilities are particularly interesting because the recycling of thismolecule reduces the amount of hydrogen peroxide that can escape fromthe sensor into the environment in which it is placed. In this context,implantable sensors that are designed to reduce the release of tissueirritants such as hydrogen peroxide will have improved biocompatibilityprofiles. Moreover as it is observed that hydrogen peroxide can reactwith enzymes such as glucose oxidase and compromise their biologicalfunction, such sensors are desired due to their avoidance of thisphenomena. Optionally, the analyte modulating layer (e.g. a glucoselimiting layer) can include compositions that serve to inhibit thediffusion of hydrogen peroxide out into the environment in which thesensor is placed. Consequently, such embodiments of the inventionimprove the biocompatibility of sensors that incorporate enzymes thatproduce hydrogen peroxide by incorporating hydrogen peroxide recyclingelements disclosed herein.

E. Permutations of Analyte Sensor Apparatus and Elements

The disclosure provided above allows artisans to generate a variety ofembodiments of the analyte sensor apparatus disclosed herein. As notedabove, illustrative general embodiments of the sensor disclosed hereininclude a base layer, a cover layer and at least one layer having asensor element such as an electrode disposed between the base and coverlayers. Typically, an exposed portion of one or more sensor elements(e.g., a working electrode, a counter electrode, reference electrode,etc.) is coated with a very thin layer of material having an appropriateelectrode chemistry. For example, an enzyme such as lactate oxidase,glucose oxidase, glucose dehydrogenase or hexokinase, can be disposed onthe exposed portion of the sensor element within an opening or aperturedefined in the cover layer. FIG. 2 illustrates a cross-section of atypical sensor structure 100 of the present invention. The sensor isformed from a plurality of layers of various conductive andnon-conductive constituents disposed on each other according to a methodof the invention to produce a sensor structure 100.

As noted above, in the sensors of the invention, the various layers(e.g. the analyte sensing layer) of the sensors can have one or morebioactive and/or inert materials incorporated therein. The term“incorporated” as used herein is meant to describe any state orcondition by which the material incorporated is held on the outersurface of or within a solid phase or supporting matrix of the layer.Thus, the material “incorporated” may, for example, be immobilized,physically entrapped, attached covalently to functional groups of thematrix layer(s). Furthermore, any process, reagents, additives, ormolecular linker agents which promote the “incorporation” of saidmaterial may be employed if these additional steps or agents are notdetrimental to, but are consistent with the objectives of the presentinvention. This definition applies, of course, to any of the embodimentsof the present invention in which a bioactive molecule (e.g. an enzymesuch as glucose oxidase) is “incorporated.” For example, certain layersof the sensors disclosed herein include a proteinaceous substance suchas albumin which serves as a crosslinkable matrix. As used herein, aproteinaceous substance is meant to encompass substances which aregenerally derived from proteins whether the actual substance is a nativeprotein, an inactivated protein, a denatured protein, a hydrolyzedspecies, or a derivatized product thereof. Examples of suitableproteinaceous materials include, but are not limited to enzymes such asglucose oxidase and lactate oxidase and the like, albumins (e.g. humanserum albumin, bovine serum albumin etc.), caseins, gamma-globulins,collagens and collagen derived products (e.g., fish gelatin, fish glue,animal gelatin, and animal glue).

An illustrative embodiment of the invention is shown in FIG. 2. Thisembodiment includes an electrically insulating base layer 102 to supportthe sensor 100. The electrically insulating layer base 102 can be madeof a material such as a ceramic substrate, which may be self-supportingor further supported by another material as is known in the art. In analternative embodiment, the electrically insulating layer 102 comprisesa polyimide substrate, for example a polyimide tape, dispensed from areel. Providing the layer 102 in this form can facilitate clean, highdensity mass production. Further, in some production processes usingsuch a polyimide tape, sensors 100 can be produced on both sides of thetape.

Typical embodiments of the invention include an analyte sensing layerdisposed on the base layer 102. In an illustrative embodiment as shownin FIG. 2 the analyte sensing layer comprises a conductive layer 104which is disposed on insulating base layer 102. Typically the conductivelayer 104 comprises one or more electrodes. The conductive layer 104 canbe applied using many known techniques and materials as will bedescribed hereafter, however, the electrical circuit of the sensor 100is typically defined by etching the disposed conductive layer 104 into adesired pattern of conductive paths. A typical electrical circuit forthe sensor 100 comprises two or more adjacent conductive paths withregions at a proximal end to form contact pads and regions at a distalend to form sensor electrodes. An electrically insulating protectivecover layer 106 such as a polymer coating is typically disposed onportions of the conductive layer 104. Acceptable polymer coatings foruse as the insulating protective layer 106 can include, but are notlimited to, non-toxic biocompatible polymers such as polyimide,biocompatible solder masks, epoxy acrylate copolymers, or the like.Further, these coatings can be photo-imageable to facilitatephotolithographic forming of apertures 108 through to the conductivelayer 104. In certain embodiments of the invention, an analyte sensinglayer is disposed upon a porous metallic and/or ceramic and/or polymericmatrix with this combination of elements functioning as an electrode inthe sensor.

In the sensors of the present invention, one or more exposed regions orapertures 108 can be made through the protective layer 106 to theconductive layer 104 to define the contact pads and electrodes of thesensor 100. In addition to photolithographic development, the apertures108 can be formed by a number of techniques, including laser ablation,chemical milling or etching or the like. A secondary photoresist canalso be applied to the cover layer 106 to define the regions of theprotective layer to be removed to form the apertures 108. An operatingsensor 100 typically includes a plurality of electrodes such as aworking electrode and a counter electrode electrically isolated fromeach other, however typically situated in close proximity to oneanother. Other embodiments may also include a reference electrode. Stillother embodiments may utilize a separate reference element not formed onthe sensor. The exposed electrodes and/or contact pads can also undergosecondary processing through the apertures 108, such as additionalplating processing, to prepare the surfaces and/or strengthen theconductive regions.

An analyte sensing layer 110 is typically disposed on one or more of theexposed electrodes of the conductive layer 104 through the apertures108. Typically, the analyte sensing layer 110 is a sensor chemistrylayer and most typically an enzyme layer. Typically, the analyte sensinglayer 110 comprises the enzyme glucose oxidase or the enzyme lactateoxidase. In such embodiments, the analyte sensing layer 110 reacts withglucose to produce hydrogen peroxide which modulates a current to theelectrode which can be monitored to measure an amount of glucosepresent. The analyte sensing layer 110 can be applied over portions ofthe conductive layer or over the entire region of the conductive layer.Typically the analyte sensing layer 110 is disposed on portions of aworking electrode and a counter electrode that comprise a conductivelayer. Some methods for generating the thin analyte sensing layer 110include spin coating processes, dip and dry processes, low shearspraying processes, ink-jet printing processes, silk screen processesand the like. Most typically the thin analyte sensing layer 110 isapplied using a spin coating process.

The analyte sensing layer 110 is typically coated with one or morecoating layers. In some embodiments of the invention, one such coatinglayer includes a membrane which can regulate the amount of analyte thatcan contact an enzyme of the analyte sensing layer. For example, acoating layer can comprise an analyte modulating membrane layer such asa glucose limiting membrane which regulates the amount of glucose thatcontacts the glucose oxidase enzyme layer on an electrode. Such glucoselimiting membranes can be made from a wide variety of materials known tobe suitable for such purposes, e.g., silicone, polyurethane, polyureacellulose acetate, Nafion, polyester sulfonic acid (Kodak AQ), hydrogelsor any other membrane known to those skilled in the art. In certainembodiments of the invention, the analyte modulating layer comprises ahydrophilic comb-copolymer having a central chain and a plurality ofside chains coupled to the central chain, wherein at least one sidechain comprises a silicone moiety.

In some embodiments of the invention, a coating layer is a glucoselimiting membrane layer 112 which is disposed above the analyte sensinglayer 110 to regulate glucose contact with the analyte sensing layer110. In some embodiments of the invention, an adhesion promoter layer114 is disposed between the membrane layer 112 and the analyte sensinglayer 110 as shown in FIG. 2 in order to facilitate their contact and/oradhesion. The adhesion promoter layer 114 can be made from any one of awide variety of materials known in the art to facilitate the bondingbetween such layers. Typically, the adhesion promoter layer 114comprises a silane compound. In alternative embodiments, protein or likemolecules in the analyte sensing layer 110 can be sufficientlycrosslinked or otherwise prepared to allow the membrane layer 112 to bedisposed in direct contact with the analyte sensing layer 110 in theabsence of an adhesion promoter layer 114.

As noted above, embodiments of the present invention can include one ormore functional coating layers. As used herein, the term “functionalcoating layer” denotes a layer that coats at least a portion of at leastone surface of a sensor, more typically substantially all of a surfaceof the sensor, and that is capable of interacting with one or moreanalytes, such as chemical compounds, cells and fragments thereof, etc.,in the environment in which the sensor is disposed. Non-limitingexamples of functional coating layers include analyte sensing layers(e.g., enzyme layers), analyte limiting layers, biocompatible layers;layers that increase the slipperiness of the sensor; layers that promotecellular attachment to the sensor; layers that reduce cellularattachment to the sensor; and the like. Typically analyte modulatinglayers operate to prevent or restrict the diffusion of one or moreanalytes, such as glucose, through the layers. Optionally such layerscan be formed to prevent or restrict the diffusion of one type ofmolecule through the layer (e.g. glucose), while at the same timeallowing or even facilitating the diffusion of other types of moleculesthrough the layer (e.g. O₂). An illustrative functional coating layer isa hydrogel such as those disclosed in U.S. Pat. Nos. 5,786,439 and5,391,250, the disclosures of each being incorporated herein byreference. The hydrogels described therein are particularly useful witha variety of implantable devices for which it is advantageous to providea surrounding water layer.

The sensor embodiments disclosed herein can include layers havingUV-absorbing polymers. In accordance with one aspect of the presentinvention, there is provided a sensor including at least one functionalcoating layer including an UV-absorbing polymer. In some embodiments,the UV-absorbing polymer is a polyurethane, a polyurea or apolyurethane/polyurea copolymer. More typically, the selectedUV-absorbing polymer is formed from a reaction mixture including adiisocyanate, at least one diol, diamine or mixture thereof, and apolyfunctional UV-absorbing monomer.

UV-absorbing polymers are used with advantage in a variety of sensorfabrication methods, such as those described in U.S. Pat. No. 5,390,671,to Lord et al., entitled “Transcutaneous Sensor Insertion Set”; No.5,165,407, to Wilson et al., entitled “Implantable Glucose Sensor”; andU.S. Pat. No. 4,890,620, to Gough, entitled “Two-Dimensional DiffusionGlucose Substrate Sensing Electrode”, which are incorporated herein intheir entireties by reference. However, any sensor production methodwhich includes the step of forming an UV-absorbing polymer layer aboveor below a sensor element is considered to be within the scope of thepresent invention. In particular, the inventive methods are not limitedto thin-film fabrication methods, and can work with other sensorfabrication methods that utilize UV-laser cutting. Embodiments can workwith thick-film, planar or cylindrical sensors and the like, and othersensor shapes requiring laser cutting.

As disclosed herein, the sensors of the present invention areparticularly designed for use as subcutaneous or transcutaneous glucosesensors for monitoring blood glucose levels in a diabetic patient.Typically each sensor comprises a plurality of sensor elements, forexample electrically conductive elements such as elongated thin filmconductors, formed between an underlying insulative thin film base layerand an overlying insulative thin film cover layer.

If desired, a plurality of different sensor elements can be included ina single sensor. For example, both conductive and reactive sensorelements can be combined in one sensor, optionally with each sensorelement being disposed on a different portion of the base layer. One ormore control elements can also be provided. In such embodiments, thesensor can have defined in its cover layer a plurality of openings orapertures. One or more openings can also be defined in the cover layerdirectly over a portion of the base layer, in order to provide forinteraction of the base layer with one or more analytes in theenvironment in which the sensor is disposed. The base and cover layerscan be comprised of a variety of materials, typically polymers. In morespecific embodiments the base and cover layers are comprised of aninsulative material such as a polyimide. Openings are typically formedin the cover layer to expose distal end electrodes and proximal endcontact pads. In a glucose monitoring application, for example, thesensor can be placed transcutaneously so that the distal end electrodesare in contact with patient blood or extracellular fluid, and thecontact pads are disposed externally for convenient connection to amonitoring device.

The sensors of the invention can have any desired configuration, forexample planar or cylindrical. The base layer 102 can beself-supportive, such as a rigid polymeric layer, or non-selfsupportive, such as a flexible film. The latter embodiment is desirablein that it permits continuous manufacture of sensors using, for example,a roll of a polymeric film which is continuously unwound and upon whichsensor elements and coating layers are continuously applied.

A general embodiment of the invention is a sensor designed forimplantation within a body that comprises a base layer, an analytesensing layer disposed upon the base layer which includes a plurality ofsensor elements, an enzyme layer (typically less than 2 microns inthickness) disposed upon the analyte sensing layer which coats all ofthe plurality of sensing elements on the conductive layer, and one ormore coating layers. Typically the enzyme layer comprises glucoseoxidase; typically in a substantially fixed ratio with a carrierprotein. In a specific embodiment, the glucose oxidase and the carrierprotein are distributed in a substantially uniform manner throughout thedisposed enzyme layer. Typically the carrier protein comprises albumin,typically in an amount of about 5% by weight. As used herein, “albumin”refers to those albumin proteins typically used by artisans to stabilizepolypeptide compositions such as human serum albumin, bovine serumalbumin and the like. In some embodiments of the invention, a coatinglayer is an analyte contacting layer which is disposed on the sensor soas to regulate the amount of analyte that can contact the enzyme layer.In further embodiments, the sensor includes an adhesion promoter layerdisposed between the enzyme layer and the analyte contacting layer; and,the enzyme layer is less than 1, 0.5, 0.25 or 0.1 microns in thickness.

F. Analyte Sensor Apparatus Configurations

In a clinical setting, accurate and relatively fast determinations ofanalytes such as glucose and/or lactate levels can be determined fromblood samples utilizing electrochemical sensors. Conventional sensorsare fabricated to be large, comprising many serviceable parts, or small,planar-type sensors which may be more convenient in many circumstances.The term “planar” as used herein refers to the well-known procedure offabricating a substantially planar structure comprising layers ofrelatively thin materials, for example, using the well-known thick orthin-film techniques. See, for example, Liu et al., U.S. Pat. No.4,571,292, and Papadakis et al., U.S. Pat. No. 4,536,274, both of whichare incorporated herein by reference. As noted below, embodiments of theinvention disclosed herein have a wider range of geometricalconfigurations (e.g. planar) than existing sensors in the art. Inaddition, certain embodiments of the invention include one or more ofthe sensors disclosed herein coupled to another apparatus such as amedication infusion pump.

FIG. 2 provides a diagrammatic view of a typical thin layer analytesensor configuration of the current invention. FIG. 3 provides adiagrammatic view of a typical thin layer analyte sensor system of thecurrent invention. Certain sensor configurations are of a relativelyflat “ribbon” type configuration that can be made with the analytesensor apparatus. Such “ribbon” type configurations illustrate anadvantage of the sensors disclosed herein that arises due to the spincoating of sensing enzymes such as glucose oxidase, a manufacturing stepthat produces extremely thin enzyme coatings that allow for the designand production of highly flexible sensor geometries. Such thin enzymecoated sensors provide further advantages such as allowing for a smallersensor area while maintaining sensor sensitivity, a highly desirablefeature for implantable devices (e.g. smaller devices are easier toimplant). Consequently, sensor embodiments of the invention that utilizevery thin analyte sensing layers that can be formed by processes such asspin coating can have a wider range of geometrical configurations (e.g.planar) than those sensors that utilize enzyme layers formed viaprocesses such as electrodeposition.

Certain sensor configurations include multiple conductive elements suchas multiple working, counter and reference electrodes. Advantages ofsuch configurations include increased surface area which provides forgreater sensor sensitivity. For example, one sensor configurationintroduces a third working sensor. One obvious advantage of such aconfiguration is signal averaging of three sensors which increasessensor accuracy. Other advantages include the ability to measuremultiple analytes. In particular, analyte sensor configurations thatinclude electrodes in this arrangement (e.g. multiple working, counterand reference electrodes) can be incorporated into multiple analytesensors. The measurement of multiple analytes such as oxygen, hydrogenperoxide, glucose, lactate, potassium, calcium, and any otherphysiologically relevant substance/analyte provides a number ofadvantages, for example the ability of such sensors to provide a linearresponse as well as ease in calibration and/or recalibration.

An exemplary multiple sensor device comprises a single device having afirst sensor which is polarized cathodically and designed to measure thechanges in oxygen concentration that occur at the working electrode (acathode) as a result of glucose interacting with glucose oxidase; and asecond sensor which is polarized anodically and designed to measurechanges in hydrogen peroxide concentration that occurs at the workingelectrode (an anode) as a result of glucose coming form the externalenvironment and interacting with glucose oxidase. As is known in theart, in such designs, the first oxygen sensor will typically experiencea decrease in current at the working electrode as oxygen contacts thesensor while the second hydrogen peroxide sensor will typicallyexperience an increase in current at the working electrode as thehydrogen peroxide generated as shown in FIG. 1 contacts the sensor. Inaddition, as is known in the art, an observation of the change incurrent that occurs at the working electrodes as compared to thereference electrodes in the respective sensor systems correlates to thechange in concentration of the oxygen and hydrogen peroxide moleculeswhich can then be correlated to the concentration of the glucose in theexternal environment (e.g. the body of the mammal).

The analyte sensors of the invention can be coupled with other medicaldevices such as medication infusion pumps. In an illustrative variationof this scheme, replaceable analyte sensors of the invention can becoupled with other medical devices such as medication infusion pumps,for example by the use of a port couple to the medical device (e.g. asubcutaneous port with a locking electrical connection).

II. Illustrative Methods and Materials for Making Analyte SensorApparatus of the Invention

A number of articles, U.S. patents and patent application describe thestate of the art with the common methods and materials disclosed hereinand further describe various elements (and methods for theirmanufacture) that can be used in the sensor designs disclosed herein.These include for example, U.S. Pat. Nos. 6,413,393; 6,368,274;5,786,439; 5,777,060; 5,391,250; 5,390,671; 5,165,407, 4,890,620,5,390,671, 5,390,691, 5,391,250, 5,482,473, 5,299,571, 5,568,806; UnitedStates Patent Application 20020090738; as well as PCT InternationalPublication Numbers WO 01/58348, WO 03/034902, WO 03/035117, WO03/035891, WO 03/023388, WO 03/022128, WO 03/022352, WO 03/023708, WO03/036255, WO03/036310 and WO 03/074107, the contents of each of whichare incorporated herein by reference.

Typical sensors for monitoring glucose concentration of diabetics arefurther described in Shichiri, et al.: “In Vivo Characteristics ofNeedle-Type Glucose Sensor-Measurements of Subcutaneous GlucoseConcentrations in Human Volunteers,” Horm. Metab. Res., Suppl. Ser.20:17-20 (1988); Bruckel, et al.: “In Vivo Measurement of SubcutaneousGlucose Concentrations with an Enzymatic Glucose Sensor and a WickMethod,” Klin. Wochenschr. 67:491-495 (1989); and Pickup, et al.: “InVivo Molecular Sensing in Diabetes Mellitus: An Implantable GlucoseSensor with Direct Electron Transfer,” Diabetologia 32:213-217 (1989).Other sensors are described in, for example Reach, et al., in ADVANCESIN IMPLANTABLE DEVICES, A. Turner (ed.), JAI Press, London, Chap. 1,(1993), incorporated herein by reference.

A. General Methods for Making Analyte Sensors

A typical embodiment of the invention disclosed herein is a method ofmaking a sensor apparatus for implantation within a mammal comprisingthe steps of: providing a base layer; forming a conductive layer on thebase layer, wherein the conductive layer includes an electrode (andtypically a working electrode, a reference electrode and a counterelectrode); forming an analyte sensing layer on the conductive layer,wherein the analyte sensing layer includes a composition that can alterthe electrical current at the electrode in the conductive layer in thepresence of an analyte; optionally forming a protein layer on theanalyte sensing layer; forming an adhesion promoting layer on theanalyte sensing layer or the optional protein layer; forming an analytemodulating layer disposed on the adhesion promoting layer, wherein theanalyte modulating layer includes a composition that modulates thediffusion of the analyte therethrough; and forming a cover layerdisposed on at least a portion of the analyte modulating layer, whereinthe cover layer further includes an aperture over at least a portion ofthe analyte modulating layer. In certain embodiments of the invention,the analyte modulating layer comprises a hydrophilic comb-copolymerhaving a central chain and a plurality of side chains coupled to thecentral chain, wherein at least one side chain comprises a siliconemoiety. In some embodiments of these methods, the analyte sensorapparatus is formed in a planar geometric configuration

As disclosed herein, the various layers of the sensor can bemanufactured to exhibit a variety of different characteristics which canbe manipulated according to the specific design of the sensor. Forexample, the adhesion promoting layer includes a compound selected forits ability to stabilize the overall sensor structure, typically asilane composition. In some embodiments of the invention, the analytesensing layer is formed by a spin coating process and is of a thicknessselected from the group consisting of less than 1, 0.5, 0.25 and 0.1microns in height.

Typically, a method of making the sensor includes the step of forming aprotein layer on the analyte sensing layer, wherein a protein within theprotein layer is an albumin selected from the group consisting of bovineserum albumin and human serum albumin. Typically, a method of making thesensor includes the step of forming an analyte sensing layer thatcomprises an enzyme composition selected from the group consisting ofglucose oxidase, glucose dehydrogenase, lactate oxidase, hexokinase andlactate dehydrogenase. In such methods, the analyte sensing layertypically comprises a carrier protein composition in a substantiallyfixed ratio with the enzyme, and the enzyme and the carrier protein aredistributed in a substantially uniform manner throughout the analytesensing layer.

B. Typical Protocols and Materials Useful in the Manufacture of AnalyteSensors

The disclosure provided herein includes sensors and sensor designs thatcan be generated using combinations of various well known techniques.The disclosure further provides methods for applying very thin enzymecoatings to these types of sensors as well as sensors produced by suchprocesses. In this context, some embodiments of the invention includemethods for making such sensors on a substrate according to art acceptedprocesses. In certain embodiments, the substrate comprises a rigid andflat structure suitable for use in photolithographic mask and etchprocesses. In this regard, the substrate typically defines an uppersurface having a high degree of uniform flatness. A polished glass platemay be used to define the smooth upper surface. Alternative substratematerials include, for example, stainless steel, aluminum, and plasticmaterials such as delrin, etc. In other embodiments, the substrate isnon-rigid and can be another layer of film or insulation that is used asa substrate, for example plastics such as polyimides and the like.

An initial step in the methods of the invention typically includes theformation of a base layer of the sensor. The base layer can be disposedon the substrate by any desired means, for example by controlled spincoating. In addition, an adhesive may be used if there is not sufficientadhesion between the substrate layer and the base layer. A base layer ofinsulative material is formed on the substrate, typically by applyingthe base layer material onto the substrate in liquid form and thereafterspinning the substrate to yield the base layer of thin, substantiallyuniform thickness. These steps are repeated to build up the base layerof sufficient thickness, followed by a sequence of photolithographicand/or chemical mask and etch steps to form the conductors discussedbelow. In an illustrative form, the base layer comprises a thin filmsheet of insulative material, such as ceramic or polyimide substrate.The base layer can comprise an alumina substrate, a polyimide substrate,a glass sheet, controlled pore glass, or a planarized plastic liquidcrystal polymer. The base layer may be derived from any materialcontaining one or more of a variety of elements including, but notlimited to, carbon, nitrogen, oxygen, silicon, sapphire, diamond,aluminum, copper, gallium, arsenic, lanthanum, neodymium, strontium,titanium, yttrium, or combinations thereof. Additionally, the substratemay be coated onto a solid support by a variety of methods well-known inthe art including physical vapor deposition, or spin-coating withmaterials such as spin glasses, chalcogenides, graphite, silicondioxide, organic synthetic polymers, and the like.

The methods of the invention further include the generation of aconductive layer having one or more sensing elements. Typically thesesensing elements are electrodes that are formed by one of the variety ofmethods known in the art such as photoresist, etching and rinsing todefine the geometry of the active electrodes. The electrodes can then bemade electrochemically active, for example by electrodeposition of Ptblack for the working and counter electrode, and silver followed bysilver chloride on the reference electrode. A sensor layer such as aanalyte sensing enzyme layer can then be disposed on the sensing layerby electrochemical deposition or a method other than electrochemicaldeposition such a spin coating, followed by vapor crosslinking, forexample with a dialdehyde (glutaraldehyde) or a carbodi-imide.

Electrodes of the invention can be formed from a wide variety ofmaterials known in the art. For example, the electrode may be made of anoble late transition metals. Metals such as gold, platinum, silver,rhodium, iridium, ruthenium, palladium, or osmium can be suitable invarious embodiments of the invention. Other compositions such as carbonor mercury can also be useful in certain sensor embodiments. Of thesemetals, silver, gold, or platinum is typically used as a referenceelectrode metal. A silver electrode which is subsequently chloridized istypically used as the reference electrode. These metals can be depositedby any means known in the art, including the plasma deposition methodcited, supra, or by an electroless method which may involve thedeposition of a metal onto a previously metallized region when thesubstrate is dipped into a solution containing a metal salt and areducing agent. The electroless method proceeds as the reducing agentdonates electrons to the conductive (metallized) surface with theconcomitant reduction of the metal salt at the conductive surface. Theresult is a layer of adsorbed metal. (For additional discussions onelectroless methods, see: Wise, E. M. Palladium: Recovery, Properties,and Uses, Academic Press, New York, N.Y. (1988); Wong, K. et al. Platingand Surface Finishing 1988, 75, 70-76; Matsuoka, M. et al. Ibid. 1988,75, 102-106; and Pearlstein, F. “Electroless Plating,” ModernElectroplating, Lowenheim, F. A., Ed., Wiley, New York, N.Y. (1974),Chapter 31). Such a metal deposition process must yield a structure withgood metal to metal adhesion and minimal surface contamination, however,to provide a catalytic metal electrode surface with a high density ofactive sites. Such a high density of active sites is a propertynecessary for the efficient redox conversion of an electroactive speciessuch as hydrogen peroxide.

In an exemplary embodiment of the invention, the base layer is initiallycoated with a thin film conductive layer by electrode deposition,surface sputtering, or other suitable process step. In one embodimentthis conductive layer may be provided as a plurality of thin filmconductive layers, such as an initial chrome-based layer suitable forchemical adhesion to a polyimide base layer followed by subsequentformation of thin film gold-based and chrome-based layers in sequence.In alternative embodiments, other electrode layer conformations ormaterials can be used. The conductive layer is then covered, inaccordance with conventional photolithographic techniques, with aselected photoresist coating, and a contact mask can be applied over thephotoresist coating for suitable photoimaging. The contact masktypically includes one or more conductor trace patterns for appropriateexposure of the photoresist coating, followed by an etch step resultingin a plurality of conductive sensor traces remaining on the base layer.In an illustrative sensor construction designed for use as asubcutaneous glucose sensor, each sensor trace can include threeparallel sensor elements corresponding with three separate electrodessuch as a working electrode, a counter electrode and a referenceelectrode.

Portions of the conductive sensor layers are typically covered by aninsulative cover layer, typically of a material such as a siliconpolymer and/or a polyimide. The insulative cover layer can be applied inany desired manner. In an exemplary procedure, the insulative coverlayer is applied in a liquid layer over the sensor traces, after whichthe substrate is spun to distribute the liquid material as a thin filmoverlying the sensor traces and extending beyond the marginal edges ofthe sensor traces in sealed contact with the base layer. This liquidmaterial can then be subjected to one or more suitable radiation and/orchemical and/or heat curing steps as are known in the art. Inalternative embodiments, the liquid material can be applied using spraytechniques or any other desired means of application. Various insulativelayer materials may be used such as photoimagable epoxyacrylate, with anillustrative material comprising a photoimagable polyimide availablefrom OCG, Inc. of West Paterson, N.J., under the product number 7020.

As noted above, appropriate electrode chemistries defining the distalend electrodes can be applied to the sensor tips, optionally subsequentto exposure of the sensor tips through the openings. In an illustrativesensor embodiment having three electrodes for use as a glucose sensor,an enzyme (typically glucose oxidase) is provided within one of theopenings, thus coating one of the sensor tips to define a workingelectrode. One or both of the other electrodes can be provided with thesame coating as the working electrode. Alternatively, the other twoelectrodes can be provided with other suitable chemistries, such asother enzymes, left uncoated, or provided with chemistries to define areference electrode and a counter electrode for the electrochemicalsensor.

Methods for producing the extremely thin enzyme coatings of theinvention include spin coating processes, dip and dry processes, lowshear spraying processes, ink jet printing processes, silk screenprocesses and the like. As artisans can readily determine the thicknessof an enzyme coat applied by process of the art, they can readilyidentify those methods capable of generating the extremely thin coatingsof the invention. Typically, such coatings are vapor crosslinkedsubsequent to their application. Surprisingly, sensors produced by theseprocesses have material properties that exceed those of sensors havingcoatings produced by electrodeposition including enhanced longevity,linearity, regularity as well as improved signal to noise ratios. Inaddition, embodiments of the invention that utilize glucose oxidasecoatings formed by such processes are designed to recycle hydrogenperoxide and improve the biocompatibility profiles of such sensors.

Sensors generated by processes such as spin coating processes also avoidother problems associated with electrodeposition, such as thosepertaining to the material stresses placed on the sensor during theelectrodeposition process. In particular, the process ofelectrodeposition is observed to produce mechanical stresses on thesensor, for example mechanical stresses that result from tensile and/orcompression forces. In certain contexts, such mechanical stresses mayresult in sensors having coatings with some tendency to crack ordelaminate. This is not observed in coatings disposed on sensor via spincoating or other low-stress processes. Consequently, yet anotherembodiment of the invention is a method of avoiding theelectrodeposition influenced cracking and/or delamination of a coatingon a sensor comprising applying the coating via a spin coating process.

Subsequent to treatment of the sensor elements, one or more additionalfunctional coatings or cover layers can then be applied by any one of awide variety of methods known in the art, such as spraying, dipping,etc. Some embodiments of the present invention include an analytemodulating layer deposited over the enzyme-containing layer. In additionto its use in modulating the amount of analyte(s) that contacts theactive sensor surface, by utilizing an analyte limiting membrane layer,the problem of sensor fouling by extraneous materials is also obviated.As is known in the art, the thickness of the analyte modulating membranelayer can influence the amount of analyte that reaches the activeenzyme. Consequently, its application is typically carried out underdefined processing conditions, and its dimensional thickness is closelycontrolled. Microfabrication of the underlying layers can be a factorwhich affects dimensional control over the analyte modulating membranelayer as well as exact the composition of the analyte limiting membranelayer material itself. In this regard, it has been discovered thatseveral types of copolymers, for example, a copolymer of a siloxane anda nonsiloxane moiety, are particularly useful. These materials can bemicrodispensed or spin-coated to a controlled thickness. Their finalarchitecture may also be designed by patterning and photolithographictechniques in conformity with the other discrete structures describedherein. Examples of these nonsiloxane-siloxane copolymers include, butare not limited to, dimethylsiloxane-alkene oxide,tetramethyldisiloxane-divinylbenzene, tetramethyldisiloxane-ethylene,dimethylsiloxane-silphenylene, dimethylsiloxane-silphenylene oxide,dimethylsiloxane-a-methylstyrene, dimethylsiloxane-bisphenol A carbonatecopolymers, or suitable combinations thereof. The percent by weight ofthe nonsiloxane component of the copolymer can be preselected to anyuseful value but typically this proportion lies in the range of about40-80 wt %. Among the copolymers listed above, thedimethylsiloxane-bisphenol A carbonate copolymer which comprises 50-55wt % of the nonsiloxane component is typical. These materials may bepurchased from Petrarch Systems, Bristol, Pa. (USA) and are described inthis company's products catalog. Other materials which may serve asanalyte limiting membrane layers include, but are not limited to,polyurethanes, cellulose acetate, cellulose nitrate, silicone rubber, orcombinations of these materials including the siloxane nonsiloxanecopolymer, where compatible.

In some embodiments of the invention, the sensor is made by methodswhich apply an analyte modulating layer that comprises a hydrophilicmembrane coating which can regulate the amount of analyte that cancontact the enzyme of the sensor layer. For example, the cover layerthat is added to the glucose sensors of the invention can comprise aglucose limiting membrane, which regulates the amount of glucose thatcontacts glucose oxidase enzyme layer on an electrode. Such glucoselimiting membranes can be made from a wide variety of materials known tobe suitable for such purposes, e.g., silicones such as polydimethylsiloxane and the like, polyurethanes, cellulose acetates, Nafion,polyester sulfonic acids (e.g. Kodak AQ), hydrogels or any othermembrane known to those skilled in the art that is suitable for suchpurposes. In certain embodiments of the invention, the analytemodulating layer comprises a hydrophilic comb-copolymer having a centralchain and a plurality of side chains coupled to the central chain,wherein at least one side chain comprises a silicone moiety. In someembodiments of the invention pertaining to sensors having hydrogenperoxide recycling capabilities, the membrane layer that is disposed onthe glucose oxidase enzyme layer functions to inhibit the release ofhydrogen peroxide into the environment in which the sensor is placed andto facilitate the contact between the hydrogen peroxide molecules andthe electrode sensing elements.

In some embodiments of the methods of invention, an adhesion promoterlayer is disposed between a cover layer (e.g. an analyte modulatingmembrane layer) and a analyte sensing layer in order to facilitate theircontact and is selected for its ability to increase the stability of thesensor apparatus. As noted herein, compositions of the adhesion promoterlayer are selected to provide a number of desirable characteristics inaddition to an ability to provide sensor stability. For example, somecompositions for use in the adhesion promoter layer are selected to playa role in interference rejection as well as to control mass transfer ofthe desired analyte. The adhesion promoter layer can be made from anyone of a wide variety of materials known in the art to facilitate thebonding between such layers and can be applied by any one of a widevariety of methods known in the art. Typically, the adhesion promoterlayer comprises a silane compound such as γ-aminopropyltrimethoxysilane.In certain embodiments of the invention, the adhesion promoting layerand/or the analyte modulating layer comprises an agent selected for itsability to crosslink a siloxane moiety present in a proximal. In otherembodiments of the invention, the adhesion promoting layer and/or theanalyte modulating layer comprises an agent selected for its ability tocrosslink an amine or carboxyl moiety of a protein present in a proximallayer. In an optional embodiment, the AP layer further comprisesPolydimethyl Siloxane (PDMS), a polymer typically present in analytemodulating layers such as a glucose limiting membrane. In illustrativeembodiments the formulation comprises 0.5-20% PDMS, typically 5-15%PDMS, and most typically 10% PDMS. The addition of PDMS to the AP layercan be advantageous in contexts where it diminishes the possibility ofholes or gaps occurring in the AP layer as the sensor is manufactured.

As noted above, a coupling reagent commonly used for promoting adhesionbetween sensor layers is γ-aminopropyltrimethoxysilane. The silanecompound is usually mixed with a suitable solvent to form a liquidmixture. The liquid mixture can then be applied or established on thewafer or planar sensing device by any number of ways including, but notlimited to, spin-coating, dip-coating, spray-coating, andmicrodispensing. The microdispensing process can be carried out as anautomated process in which microspots of material are dispensed atmultiple preselected areas of the device. In addition, photolithographictechniques such as “lift-off” or using a photoresist cap may be used tolocalize and define the geometry of the resulting permselective film(i.e. a film having a selective permeability). Solvents suitable for usein forming the silane mixtures include aqueous as well as water-miscibleorganic solvents, and mixtures thereof. Alcoholic water-miscible organicsolvents and aqueous mixtures thereof are particularly useful. Thesesolvent mixtures may further comprise nonionic surfactants, such aspolyethylene glycols (PEG) having a for example a molecular weight inthe range of about 200 to about 6,000. The addition of these surfactantsto the liquid mixtures, at a concentration of about 0.005 to about 0.2g/dL of the mixture, aids in planarizing the resulting thin films. Also,plasma treatment of the wafer surface prior to the application of thesilane reagent can provide a modified surface which promotes a moreplanar established layer. Water-immiscible organic solvents may also beused in preparing solutions of the silane compound. Examples of theseorganic solvents include, but are not limited to, diphenylether,benzene, toluene, methylene chloride, dichloroethane, trichloroethane,tetrachloroethane, chlorobenzene, dichlorobenzene, or mixtures thereof.When protic solvents or mixtures thereof are used, the water eventuallycauses hydrolysis of the alkoxy groups to yield organosilicon hydroxides(especially when n=1) which condense to form poly(organosiloxanes).These hydrolyzed silane reagents are also able to condense with polargroups, such as hydroxyls, which may be present on the substratesurface. When aprotic solvents are used, atmospheric moisture may besufficient to hydrolyze the alkoxy groups present initially on thesilane reagent. The R′ group of the silane compound (where n=1 or 2) ischosen to be functionally compatible with the additional layers whichare subsequently applied. The R′ group usually contains a terminal aminegroup useful for the covalent attachment of an enzyme to the substratesurface (a compound, such as glutaraldehyde, for example, may be used asa linking agent as described by Murakami, T. et al., Analytical Letters1986, 19, 1973-86).

Like certain other coating layers of the sensor, the adhesion promoterlayer can be subjected to one or more suitable radiation and/or chemicaland/or heat curing steps as are known in the art. In alternativeembodiments, the enzyme layer can be sufficiently crosslinked orotherwise prepared to allow the membrane cover layer to be disposed indirect contact with the analyte sensing layer in the absence of anadhesion promoter layer.

An illustrative embodiment of the invention is a method of making asensor by providing a base layer, forming a sensor layer on the baselayer, spin coating an enzyme layer on the sensor layer and then formingan analyte contacting layer (e.g. an analyte modulating layer such as aglucose limiting membrane) on the sensor, wherein the analyte contactinglayer regulates the amount of analyte that can contact the enzyme layer.In some methods, the enzyme layer is vapor crosslinked on the sensorlayer. In a typical embodiment of the invention, the sensor layer isformed to include at least one working electrode and at least onecounter electrode. In certain embodiments, the enzyme layer is formed onat least a portion of the working electrode and at least a portion ofthe counter electrode. Typically, the enzyme layer that is formed on thesensor layer is less than 2, 1, 0.5, 0.25 or 0.1 microns in thickness.Typically, the enzyme layer comprises one or more enzymes such asglucose oxidase, glucose dehydrogenase, lactate oxidase, hexokinase orlactate dehydrogenase and/or like enzymes. In a specific method, theenzyme layer comprises glucose oxidase that is stabilized by coating iton the sensor layer in combination with a carrier protein in a fixedratio. Typically the carrier protein is albumin. Typically such methodsinclude the step of forming an adhesion promoter layer disposed betweenthe glucose oxidase layer and the analyte contacting layer. Optionally,the adhesion promoter layer is subjected to a curing process prior tothe formation of the analyte contacting layer.

A related embodiment of the invention is a method of making a glucosesensor by providing a base layer, forming a sensor layer on the baselayer that includes at least one working electrode and at least onecounter electrode, forming a glucose oxidase layer on the sensor layerby a spin coating process (a layer which is typically stabilized bycombining the glucose oxidase with albumin in a fixed ratio), whereinthe glucose oxidase layer coats at least a portion of the workingelectrode and at least a portion of the counter electrode, and thenforming a glucose limiting layer on the glucose sensor so as to regulatethe amount of glucose that can contact the glucose oxidase layer. Insuch processes, the glucose oxidase layer that is formed on the sensorlayer is typically less than 2, 1, 0.5, 0.25 or 0.1 microns inthickness. Typically, the glucose oxidase coating is vapor crosslinkedon the sensor layer. Optionally, the glucose oxidase coating covers theentire sensor layer. In certain embodiments of the invention, anadhesion promoter layer is disposed between the glucose oxidase layerand the analyte contacting layer. In certain embodiments of theinvention, the analyte sensor further comprises one or more cover layerswhich are typically electrically insulating protective layers (see, e.g.element 106 in FIG. 2). Typically, such cover layers are disposed on atleast a portion of the analyte modulating layer.

The finished sensors produced by such processes are typically quicklyand easily removed from a supporting substrate (if one is used), forexample, by cutting along a line surrounding each sensor on thesubstrate. The cutting step can use methods typically used in this artsuch as those that include a UV laser cutting device that is used to cutthrough the base and cover layers and the functional coating layersalong a line surrounding or circumscribing each sensor, typically in atleast slight outward spaced relation from the conductive elements sothat the sufficient interconnected base and cover layer material remainsto seal the side edges of the finished sensor. In addition, dicingtechniques typically used to cut ceramic substrates can be used with theappropriate sensor embodiments. Since the base layer is typically notphysically attached or only minimally adhered directly to the underlyingsupporting substrate, the sensors can be lifted quickly and easily fromthe supporting substrate, without significant further processing stepsor potential damage due to stresses incurred by physically pulling orpeeling attached sensors from the supporting substrate. The supportingsubstrate can thereafter be cleaned and reused, or otherwise discarded.The functional coating layer(s) can be applied either before or afterother sensor components are removed from the supporting substrate (e.g.,by cutting).

III. Methods for Using Analyte Sensor Apparatus of the Invention

Related embodiments of the invention is a method of sensing an analytewithin the body of a mammal, the method comprising implanting an analytesensor embodiment disclosed herein in to the mammal and then sensing oneor more electrical fluctuations such as alteration in current at theworking electrode and correlating the alteration in current with thepresence of the analyte, so that the analyte is sensed. Typically theanalyte sensor is polarized anodically such that the working electrodewhere the alteration in current is sensed is an anode. In one suchmethod, the analyte sensor apparatus senses glucose in the mammal. In analternative method, the analyte sensor apparatus senses lactate,potassium, calcium, oxygen, pH, and/or any physiologically relevantanalyte in the mammal.

Certain analyte sensors having the structure discussed above have anumber of highly desirable characteristics which allow for a variety ofmethods for sensing analytes in a mammal. For example in such methods,the analyte sensor apparatus implanted in the mammal functions to sensean analyte within the body of a mammal for more than 1, 2, 3, 4, 5, or 6months. Typically, the analyte sensor apparatus so implanted in themammal senses an alteration in current in response to an analyte within15, 10, 5 or 2 minutes of the analyte contacting the sensor. In suchmethods, the sensors can be implanted into a variety of locations withinthe body of the mammal, for example in both vascular and non-vascularspaces.

IV. Kits and Sensor Sets of the Invention

In another embodiment of the invention, a kit and/or sensor set, usefulfor the sensing an analyte as is described above, is provided. The kitand/or sensor set typically comprises a container, a label and ananalyte sensor as described above. Suitable containers include, forexample, an easy to open package made from a material such as a metalfoil, bottles, vials, syringes, and test tubes. The containers may beformed from a variety of materials such as metals (e.g. foils) paperproducts, glass or plastic. The label on, or associated with, thecontainer indicates that the sensor is used for assaying the analyte ofchoice. In some embodiments, the container holds a porous matrix that iscoated with a layer of an enzyme such as glucose oxidase. The kit and/orsensor set may further include other materials desirable from acommercial and user standpoint, including elements or devices designedto facilitate the introduction of the sensor into the analyteenvironment, other buffers, diluents, filters, needles, syringes, andpackage inserts with instructions for use.

Various publication citations are referenced throughout thespecification. In addition, certain text from related art is reproducedherein to more clearly delineate the various embodiments of theinvention. The disclosures of all citations in the specification areexpressly incorporated herein by reference.

The invention claimed is:
 1. An analyte sensing system, the analytesensing system comprising: an analyte sensor for implantation in amammal; and a sensor electronics device in communication with theanalyte sensor, the sensor electronics device including circuitry to:apply a first electrode potential to an analyte sensor for a firstpredetermined period of time of at least 5 seconds; apply a secondelectrode potential to the analyte sensor for a second predeterminedperiod of time of at least 5 seconds; repeat the application of thefirst electrode potential and the second electrode potentialcontinuously over a sensor duration time period of greater than 3 hours;receive signals from the analyte sensor during the sensor duration timeperiod; and calculate a concentration of the analyte from the signalsreceived and monitored from the analyte sensor; wherein calculating theconcentration of analyte includes analyzing relaxation kinetics of thesignals from the analyte sensor during the first predetermined period oftime or the second predetermined period of time.
 2. The analyte sensingsystem of claim 1, wherein the sensor electronics device furtherincludes circuitry to initiate the analyte sensor prior to theapplication of the first electrode potential for the first predeterminedperiod of time.
 3. The analyte sensing system of claim 1, wherein thesensor electronics device further includes circuitry to apply a thirdelectrode potential to the analyte sensor for a third predeterminedperiod of time and repeat the applicant of the first electrodepotential, second electrode potential and third electrode potential overthe sensor duration time period.
 4. The analyte sensing system of claim3, wherein the first, second and third electrode potential are steppedelectrode potentials.
 5. The analyte sensing system of claim 1, furthercomprising a monitoring device in communication with the electronicsdevice, wherein the monitoring device includes circuitry to monitor thesignals received from the analyte sensor and to calculate theconcentration of the analyte from the signals.
 6. The analyte sensingsystem of claim 5, wherein calculating the concentration of analyteincludes evaluating the overall change in the signals from the analytesensor during the first predetermined period of time.
 7. The analytesensing system of claim 5, wherein the concentration of analyte includescalculating the total charge transfer from signals received from theanalyte sensor during the first predetermined period of time.
 8. Theanalyte sensing system of claim 1, wherein calculating the concentrationof analyte further includes calculating current as a function of timeusing an equation:${i(t)} = \frac{{nFAD}^{1\text{/}2}C_{0}}{\pi^{1\text{/}2}t^{1\text{/}2}}$where i is current, t is time, n is number of electrons involved in thereaction; F is Faraday's constant, A is electrode surface area (cm²), C₀is concentration of electroactive species (mol/cm³), and D is diffusionconstant for electroactive species (cm²/s).